Human antibodies to influenza hemagglutinin

ABSTRACT

The present invention provides monoclonal antibodies, or antigen-binding fragments thereof, that bind to the influenza hemagglutinin (HA) protein, pharmaceutical compositions comprising the antibodies and methods of use. The antibodies of the invention are useful for inhibiting or neutralizing influenza virus activity, thus providing a means of treating or preventing influenza infection in humans. In some embodiments, the invention provides for use of one or more antibodies that bind to the influenza HA for preventing viral attachment and/or entry into host cells. The antibodies of the invention may be used prophylactically or therapeutically and may be used alone or in combination with one or more other anti-viral agents or vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patent application Ser. No. 14/974,361, filed Dec. 18, 2015, which claims the benefit under 35 U.S.C § 119(e) of U.S. provisional application Nos. 62/094,752, filed Dec. 19, 2014 and 62/152,122 filed Apr. 24, 2015, all of which are herein specifically incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related to human antibodies and antigen-binding fragments thereof that specifically bind to influenza hemagglutinin (HA), compositions comprising these antibodies and therapeutic and diagnostic methods of using these antibodies.

SEQUENCE LISTING

An official copy of the sequence listing is submitted concurrently with the present specification electronically via EFS-Web as an ASCII formatted sequence listing with a file name of “10119US02_SEQ_LIST_ST25”, a creation date of Jul. 25, 2019, and a size of about 2.11 MB. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Influenza is a highly contagious disease, which has a long history characterized by waves of pandemics, epidemics, resurgences and outbreaks. In spite of annual vaccination efforts, influenza infections result in substantial morbidity and mortality.

Influenza viruses consist of three types, A, B and C. Furthermore, influenza A viruses can be classified into subtypes based on allelic variations in antigenic regions of two genes that encode the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and entry into the host cell.

Hemagglutinin is a trimeric glycoprotein that contains two structural domains, a globular head domain that consists of the receptor-binding site (that is subject to frequent antigenic drift) and the stem region (more conserved among various strains of influenza virus). The HA protein is synthesized as a precursor (HA0), which undergoes proteolytic processing to produce two subunits (HA1 and HA2), which associate with one another to form the stem/globular head structure. The HA1 peptide is responsible for the attachment of virus to the cell surface. The HA2 peptide forms a stem-like structure that mediates the fusion of viral and cell membranes in endosomes, allowing the release of the ribonucleoprotein complex into the cytoplasm.

Currently, there are eighteen subtypes defined by their hemagglutinin proteins (H1-H18). The 18 HAs can be classified into two groups. Group 1 consists of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18 subtypes, and group 2 includes H3, H4, H7, H10, H14 and H15 subtypes.

New strains of the same subtype may arise as a result of a phenomenon called antigenic drift, or mutations in the HA or NA molecules which generate new and different epitopes. A consequence of this is that a new vaccine must be produced every year against viruses that are predicted to emerge, a process that is not only costly, but highly inefficient. While technological advances have improved the ability to produce improved influenza antigen(s) for vaccine compositions, there remains a need to provide additional sources of protection to address emerging subtypes and strains of influenza.

While the idea of a vaccine composition comprising the antigen of interest (e.g. the HA and/or NA) to generate broadly neutralizing antibodies in a patient is generally thought to be a good approach, it is not always desirable to use this approach in certain patient populations. For example, in certain patients, a vaccine composition comprising the antigen of interest may not always be effective, such as in the elderly, in the very young, in immunocompromised patients, etc. In these patient populations, or in any patient who is not able to mount an effective immune response, it may be more beneficial to provide a composition already containing broadly neutralizing antibodies that may target epitopes common to a variety of strains within Group 1 and/or Group 2 subtypes.

To date there has been limited success in identifying such antibodies that broadly neutralize or inhibit influenza viruses. Okuno et al. immunized mice with influenza A/Okuda/57 (H2N2) and isolated an antibody designated C179, which bound to a conserved conformational epitope in HA2 and neutralized the Group 1 H2, H1 and H5 subtype influenza A viruses in vitro and in vivo (Okuno et al. (1993) J. Virol. 67(5):2552-2558). Throsby et al. identified 13 monoclonal antibodies from human B cells that had broad activity against Group 1 subtypes (Throsby et al. (2008), PLOS one 3(2):e3942). Sui et al. identified a human monoclonal antibody (F10), which bound H5 and other Group 1 viruses (Sui, et al. (2009), Nat. Struct. Mol. Biol. 16(3):265-273).

However, after decades of research in this area, only a few antibodies are currently in clinical trials to assess their ability to neutralize influenza viruses of different subtypes (See, for example, antibodies under development by Crucell Holland ((US2012/0276115, US2014/0065156, U.S. Pat. No. 8,470,327, US2014/0120113, EP2731967, U.S. Pat. No. 8,691,223, US2013/0243792, US2014/0065165, WO2008/028946 and WO2010/130636); Osaka University (US2011/0319600, EP2380976, US2012/0058124, US2012/0058124), Celltrion (US2013/0004505, EP2545074; WO2014/158001); Vanderbilt University (US2013/0289246), SeaLane Biotechnologies (US2012/0128671), Trellis Bioscience, Inc. (US2012/0020971 EP2582721); Visterra, Inc. (US2013/0302349); Burnham Institute/Dana Farber (US2014/011982, EP2222701, WO2010/027818); Temasek (U.S. Pat. Nos. 8,444,986, 8,574,581, 8,637,644, 8,637,645, 8,383,121, 8,540,996, 8,574,830, 8,540,995); HUMABS Biosciences/Institute for Research in Biomedicine (U.S. Pat. No. 8,871,207); MedImmune (WO2015/051010); and Genentech (US2014/0161822), but there are still no marketed antibodies that broadly neutralize or inhibit influenza A virus infection or attenuate the disease caused by various subtypes of this virus. Accordingly, there is still a need in the art to identify new antibodies that neutralize multiple subtypes of influenza A virus, which can be used to prevent or treat an influenza virus infection.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen-binding fragments thereof that bind influenza hemagglutinin (HA). The antibodies of the present invention are useful, inter alia, for inhibiting or neutralizing the activity of influenza HA. In some embodiments, the antibodies are useful for blocking attachment of the influenza virus to the host cell and/or for preventing the entry of the influenza virus into host cells. In some embodiments, the antibodies function by inhibiting the cell-to-cell transmission of the virus. In certain embodiments, the antibodies are useful in preventing, treating or ameliorating at least one symptom of influenza virus infection in a subject. In certain embodiments, the antibodies may be administered prophylactically or therapeutically to a subject having, or at risk of acquiring, an influenza virus infection. In certain embodiments, compositions containing at least one antibody of the invention may be administered to a subject for whom a vaccine is contra-indicated, or for whom a vaccine is less efficacious, for example, an elderly patient, a very young patient, a patient who may be allergic to any one or more components of a vaccine, or an immunocompromised patient who may be non-responsive to the immunogens in a vaccine. In certain embodiments, compositions containing at least one antibody of the invention may be administered to medical staff, hospitalized patients or nursing home residents or other high-risk patients during an influenza outbreak. In certain embodiments, compositions containing at least one antibody of the invention may be administered as a first line treatment to patients in the event that a predicted yearly vaccine is ineffective, or in the event of a pandemic with a strain that has undergone a major antigenic shift.

The antibodies of the invention can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab′)2 or scFv fragment), and may be modified to affect functionality, e.g., to increase persistence in the host or to increase effector function or eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibodies may be bispecific.

In a first aspect, the present invention provides isolated recombinant monoclonal antibodies or antigen-binding fragments thereof that bind specifically to the influenza HA.

In one embodiment, the present invention provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza A hemagglutinin (HA), wherein the antibody has two or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds to influenza HA with a dissociation constant (K_(D)) of less than 10⁻⁹M, as measured in a real-time bio-layer interferometer based biosensor (Octet HTX assay);

(c) demonstrates a dissociative half-life (t½) greater than 370 minutes;

(d) demonstrates neutralization of group 1 influenza A viruses selected from H1N1, H5N1, H9N2, H13N6 and H16N3, with an 10₅₀ of less than 130 nM;

(e) demonstrates complement mediated lysis of influenza virus infected cells with an EC₅₀ of less than 66 nM;

(f) demonstrates protection, as measured by increased survival in an animal model of influenza virus infection when administered either before or after virus challenge; or

(g) wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences listed in Table 1 or Table 12; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) sequences listed in Table 1 or Table 12.

In one embodiment, an antibody of the invention demonstrates a dissociative half-life in monkeys that is about 1.5 fold greater than a comparator antibody designated Control I mAb and a dissociative half-life in mice that is about 2 fold greater than the Control I mAb.

In certain embodiments, an antibody of the invention demonstrates an increase in protection as compared to oseltamivir, when administered at 48 hours post infection, or at 72 hours post infection to a mammal infected with influenza virus.

In a related embodiment, an antibody of the invention confers an increase in protection in a mammal infected with influenza virus when administered either subcutaneously or intravenously and/or when administered prior to infection, or after infection with influenza virus.

In one embodiment, an antibody of the invention demonstrates an increase in protection, as compared to an animal administered an isotype (negative) control antibody, when administered to an infected mammal as a single subcutaneous or intravenous dose ranging from about 0.01 mg/kg to about 50 mg/kg.

In one embodiment, an antibody of the invention demonstrates an increase in protection when administered to an influenza virus infected mammal as a single intravenous dose of about 15 mg/kg compared to oral administration of oseltamivir administered twice daily for 5 days at a dose of about 5 mg/kg to about 25 mg/kg.

In one embodiment, an antibody of the invention demonstrates a survival rate of greater than about 20% in a mammal infected with influenza virus, when administered prophylactically as a single subcutaneous dose ranging from about 0.01 mg/kg to about 2 mg/kg.

In one embodiment, an antibody of the invention demonstrates a survival rate of greater than about 30% in a mammal infected with influenza virus, when administered as a single intravenous dose ranging from about 7 mg/kg to about 50 mg/kg by at least 24 hours post infection.

In one embodiment, an antibody of the invention demonstrates a survival rate of about 30% to about 60% in a mammal infected with influenza virus, when administered as a single intravenous dose of about 7 mg/kg to about 50 mg/kg when administered at 48 hours post infection.

In one embodiment, an antibody of the invention demonstrates a survival rate of equal to, or greater than about 60% in a mammal infected with influenza virus, when administered as a single intravenous dose of about 15 mg/kg to about 30 mg/kg at 48 hours or longer post infection.

In one embodiment, an antibody of the invention demonstrates a survival rate of about 100% in a mammal infected with influenza virus, when administered as a single intravenous dose of about 15 mg/kg at 48 hours or longer post infection.

In one embodiment, an antibody of the invention demonstrates a survival rate of about 100% in a mammal infected with influenza virus, when administered as a single intravenous dose of about 15 mg/kg compared to a 40% survival rate observed with oseltamivir when administered orally twice a day for 5 days at a dose of about 25 mg/kg.

In one embodiment, an antibody of the invention provides an additive protective effect in a mammal infected with influenza virus when administered with oseltamivir at greater than 48 hours post infection.

In one embodiment, an antibody of the invention provides an additive protective effect in a mammal infected with influenza virus when administered with oseltamivir at 72 hours post infection.

In one embodiment, an antibody of the invention provides an additive protective effect when used in combination with oseltamivir when the antibody is administered to an influenza virus infected mammal as a single intravenous dose ranging from about 7 mg/kg to about 15 mg/kg and the oseltamivir is administered orally twice daily for 5 days at a dose of about 25 mg/kg.

In a related embodiment, an antibody of the invention provides an additive protective effect when used in combination with oseltamivir at 72 hours after influenza virus infection, wherein the antibody is administered as a single intravenous dose ranging from about 7 mg/kg to about 15 mg/kg and the oseltamivir is administered orally twice daily for 5 days at a dose of about 25 mg/kg.

In one embodiment, an antibody of the invention may be administered intravenously, intranasally, subcutaneously, intradermally, or intramuscularly and the oseltamivir may be administered orally.

In one embodiment, the oseltamivir is administered prior to, concurrently with, or after administration of an antibody of the invention.

In one embodiment, the antibody and/or the oseltamivir may be administered as a single dose, or as multiple doses.

Exemplary anti-influenza HA antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of exemplary anti-influenza HA antibodies. Table 2 sets forth the nucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-influenza HA antibodies.

Further exemplary anti-influenza HA antibodies of the present invention are listed in Tables 12 and 13 herein. Table 12 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of exemplary anti-influenza HA antibodies. Table 13 sets forth the nucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary anti-influenza HA antibodies.

The present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

In one embodiment the invention provides antibodies, or antigen-binding fragments thereof, which specifically bind influenza HA, comprising a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 242, 250, 258, 266, 274, 282 and 290.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

In one embodiment the invention provides antibodies, or antigen-binding fragments thereof, which specifically bind influenza HA, comprising a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210 and 226.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1, or Table 12 paired with any of the LCVR amino acid sequences listed in Table 1, or Table 12. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-influenza HA antibodies listed in Table 1, or Table 12.

In one embodiment, the isolated antibody or antigen-binding fragment that specifically binds influenza HA comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 50/66, 74/82, 74/66, 90/98, 106/114, 122/130, 138/146, 154/162, 170/178, 186/194, 202/210, 218/226, 234/66, 242/66, 250/66, 258/66, 266/66, 274/66, 282/66 and 290/66.

In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 18/26 (e.g., H1H11729P), 50/58 (e.g., H1H11829N), 50/66 (e.g. H1H11829N2), or 106/114 (e.g., H1H14571N).

In one embodiment, the isolated antibody or antigen-binding fragment comprises:

(a) a HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 244, 252, 260, 268, 276, 284, and 292;

(b) a HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 246, 254, 262, 270, 278, 286, and 294;

(c) a HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 248, 256, 264, 272, 280, 288, and 296;

(d) a LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, and 228;

(e) a LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, and 230; and

(f) a LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216 and 232.

In one embodiment, the isolated antibody or antigen-binding fragment, which specifically binds influenza HA, comprises (a) a HCDR1 of SEQ ID NO: 20, (b) a HCDR2 of SEQ ID NO: 22; (c) a HCDR3 of SEQ ID NO: 24; (d) a LCDR1 of SEQ ID NO: 28; (e) a LCDR2 of SEQ ID NO: 30 and (f) a LCDR3 of SEQ ID NO: 32.

In one embodiment, the isolated antibody or antigen-binding fragment, which specifically binds influenza HA, comprises (a) a HCDR1 of SEQ ID NO: 52, (b) a HCDR2 of SEQ ID NO: 54; (c) a HCDR3 of SEQ ID NO: 56; (d) a LCDR1 of SEQ ID NO: 68; (e) a LCDR2 of SEQ ID NO: 70 and (f) a LCDR3 of SEQ ID NO: 72.

In one embodiment, the isolated antibody or antigen-binding fragment, which specifically binds influenza HA, comprises (a) a HCDR1 of SEQ ID NO: 52, (b) a HCDR2 of SEQ ID NO: 54; (c) a HCDR3 of SEQ ID NO: 56; (d) a LCDR1 of SEQ ID NO: 60; (e) a LCDR2 of SEQ ID NO: 62 and (f) a LCDR3 of SEQ ID NO: 64.

In one embodiment, the isolated antibody or antigen-binding fragment, which specifically binds influenza HA, comprises (a) a HCDR1 of SEQ ID NO: 108, (b) a HCDR2 of SEQ ID NO: 110; (c) a HCDR3 of SEQ ID NO: 112; (d) a LCDR1 of SEQ ID NO: 116; (e) a LCDR2 of SEQ ID NO: 118 and (f) a LCDR3 of SEQ ID NO: 120.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or Table 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1, or Table 12, paired with any of the LCDR3 amino acid sequences listed in Table 1, or Table 12. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-influenza HA antibodies listed in Table 1, or Table 12. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 24/32 (e.g., H1H11729P), 56/64 (e.g., H1H11829N), 56/72 (e.g. H1H11829N2) and 112/120 (e.g., H1H14571N).

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-influenza HA antibodies listed in Table 1, or Table 12. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 20-22-24-28-30-32 (e.g., H1H11729P), 52-54-56-60-62-64 (e.g., H1H11829N); 52-54-56-68-70-72 (e.g. H1H11829N2) and 108-110-112-116-118 and 120 (e.g., H1H14571N).

In a related embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-influenza HA antibodies listed in Table 1, or Table 12. For example, the present invention includes antibodies, or antigen-binding fragments thereof, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18/26 (e.g., H1H11729P), 50/58 (e.g., H1H11829N), 50/66 (e.g. H1H11829N2) and 106/114 (e.g., H1H14571N). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

The present invention includes anti-influenza HA antibodies having a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

The present invention also provides for antibodies and antigen-binding fragments thereof that compete for specific binding to influenza HA with an antibody or antigen-binding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1, or Table 12.

The present invention also provides antibodies and antigen-binding fragments thereof that cross-compete for binding to influenza HA, or that bind the same epitope on influenza HA, as a reference antibody or antigen-binding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1, or Table 12.

The present invention also provides isolated antibodies and antigen-binding fragments thereof that block influenza HA attachment to, and/or entry into a host cell.

In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific comprising a first binding specificity to a first epitope in the influenza HA and a second binding specificity to another antigen.

In a second aspect, the present invention provides nucleic acid molecules encoding anti-influenza HA antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, or Table 12; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-influenza HA antibodies listed in Table 1, or Table 12.

The present invention also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary anti-influenza HA antibodies listed in Table 1, or Table 12.

The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, or Table 12 and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1, or Table 12. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or Table 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-influenza HA antibody listed in Table 1, or Table 12.

The present invention provides nucleic acid molecules encoding any of the heavy chain amino acid sequences listed in Table 1, or Table 12. The present invention also provides nucleic acid molecules encoding any of the light chain amino acid sequences listed in Table 1, or Table 12.

In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-influenza HA antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as set forth in Table 1, or Table 12. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

In a third aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen-binding fragment thereof which specifically binds influenza HA and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition, which is a combination of an anti-influenza HA antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-influenza HA antibody. Exemplary agents that may be advantageously combined with an anti-influenza HA antibody include, without limitation, other agents that bind and/or inhibit influenza HA activity (including other antibodies or antigen-binding fragments thereof, etc.) and/or agents, which do not directly bind influenza HA but nonetheless inhibit viral activity including infectivity of host cells. In certain embodiments, the invention provides for a pharmaceutical composition comprising: (a) a first anti-influenza HA antibody or antigen-binding fragment thereof; (b) a second anti-influenza HA antibody or antigen-binding fragment thereof, wherein the first antibody binds to a first epitope on influenza HA and the second antibody binds to a second epitope on influenza HA wherein the first and second epitopes are distinct and non-overlapping; and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the invention provides for a pharmaceutical composition comprising: (a) a first anti-influenza HA antibody or antigen-binding fragment thereof; (b) a second anti-influenza HA antibody or antigen-binding fragment thereof, wherein the first antibody does not cross-compete with the second antibody for binding to influenza HA; and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the invention provides for a pharmaceutical composition comprising: (a) a first anti-influenza HA antibody or antigen-binding fragment thereof; (b) a second anti-influenza antibody or antigen-binding fragment thereof, which interacts with a different influenza antigen, wherein the first antibody binds to an epitope on influenza HA and the second antibody binds to an epitope on a different influenza antigen; and (c) a pharmaceutically acceptable carrier or diluent. In certain embodiments, the invention provides for a pharmaceutical composition comprising: (a) a first anti-influenza HA antibody or antigen-binding fragment thereof; (b) a second antibody or antigen-binding fragment thereof, which interacts with a different viral (non-influenza) antigen, wherein the first antibody binds to an epitope on influenza HA and the second antibody binds to an epitope on a different viral (non-influenza) antigen; and (c) a pharmaceutically acceptable carrier or diluent. Additional combination therapies and co-formulations involving the anti-influenza HA antibodies of the present invention are disclosed elsewhere herein.

In a fourth aspect, the invention provides therapeutic methods for treating a disease or disorder associated with influenza HA (such as viral infection in a subject), or at least one symptom associated with the viral infection, using an anti-influenza HA antibody or antigen-binding portion of an antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention to the subject in need thereof. The disorder treated is any disease or condition, which is improved, ameliorated, inhibited or prevented by inhibition of influenza HA activity. In certain embodiments, the invention provides methods to prevent, treat or ameliorate at least one symptom of influenza A infection, the method comprising administering a therapeutically effective amount of an anti-influenza HA antibody or antigen-binding fragment thereof of the invention to a subject in need thereof.

In some embodiments, the present invention provides methods to ameliorate or reduce the severity, duration, or frequency of occurrence, of at least one symptom of influenza infection in a subject by administering an anti-influenza HA antibody of the invention, wherein the at least one symptom is selected from the group consisting of headache, fever, aches, rhinorrhea (nasal congestion), chills, fatigue, weakness, sore throat, cough, shortness of breath, vomiting, diarrhea, pneumonia, bronchitis, and death.

In certain embodiments, the invention provides methods to decrease viral load in a subject, the methods comprising administering to the subject an effective amount of an antibody or fragment thereof of the invention that binds influenza HA and blocks influenza HA binding and/or entry into the host cell.

In certain embodiments, the antibody or antigen-binding fragment thereof may be administered prophylactically or therapeutically to a subject having, or at risk of having, or pre-disposed to developing an influenza infection. The subjects at risk include, but are not limited to, an immunocompromised person, for example, a person who is immunocompromised because of autoimmune disease, or those persons receiving immunosuppressive therapy (for example, following organ transplant), or those persons afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), certain forms of anemia that deplete or destroy white blood cells, those persons receiving radiation or chemotherapy, or those persons afflicted with an inflammatory disorder. Other subjects at risk for acquiring an influenza infection include an elderly adult (more than 65 years of age), children younger than 2 years of age, healthcare workers, and people with underlying medical conditions such as pulmonary infection, heart disease or diabetes. Also, any person who comes into physical contact or close physical proximity with an infected individual has an increased risk of developing an influenza virus infection. Moreover, a subject is at risk of contracting an influenza infection due to proximity to an outbreak of the disease, e.g. subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of influenza virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.

In certain embodiments, the antibody or antigen-binding fragment thereof of the invention is administered in combination with a second therapeutic agent to the subject in need thereof. The second therapeutic agent may be selected from the group consisting of an anti-inflammatory drug (such as corticosteroids, and non-steroidal anti-inflammatory drugs), an anti-infective drug, a different antibody to influenza HA, an antibody to a different influenza antigen (e.g. the neuraminidase), an anti-viral drug, a decongestant, an anti-histamine, a vaccine for influenza, a dietary supplement such as anti-oxidants and any other drug or therapy known in the art useful for ameliorating at least one symptom of the influenza infection, or for reducing the viral load in a patient. In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effect(s) associated with an antibody or antigen-binding fragment thereof of the invention, if such side effect(s) should occur. The antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intranasally, intramuscularly, or intracranially. In one embodiment, the antibody may be administered as a single intravenous infusion for maximum concentration of the antibody in the serum of the subject. The antibody or fragment thereof may be administered at a dose of about 0.01 mg/kg of body weight to about 100 mg/kg of body weight of the subject. In certain embodiments, an antibody of the present invention may be administered at one or more doses comprising between 50 mg to 5000 mg.

The present invention also includes use of an anti-influenza HA antibody or antigen-binding fragment thereof of the invention for treating a disease or disorder that would benefit from the blockade of influenza HA binding and/or activity. The present invention also includes use of an anti-influenza HA antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from the blockade of influenza HA binding and/or activity.

Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Shows that a single dose of H1H11729P at 48 hrs post infection demonstrates greater efficacy than oseltamivir at 48 hrs post infection in treating severe influenza A virus infection in mice. A single dose of H1H11729P at 15 mg/kg (circles) given 48 h p.i. is more efficacious than oseltamivir (TAMIFLU®) dosed twice daily (BID) for 5 days starting on day 2 post infection at 25 (inverse triangles) or 5 mg/kg (diamonds). Mice were infected intranasally (IN) on day 0 with 10×MLD₅₀ of A/Puerto Rico/08/1934 (H1N1). Control groups included an uninfected (triangles, dotted line) and infected (hexagons) group that received oral gavage of water and CR8020 as an IgG1 isotype (negative) control.

FIG. 2. Shows that additive efficacy is observed when a single dose of H1H11729P is combined with oseltamivir at 72 hrs post infection to treat severe influenza in mice. Mice received a single sub-efficacious dose of 7 (squares, dotted line) or 15 mg/kg of H1H11729P (circles, dotted line), control IgG (triangles), 25 mg/kg BID oseltamivir for 5 days (diamonds, dotted line) or a combination of a single dose of 7 (squares, solid line) or 15 mg/kg H1H11729P (circles, solid line) and the regimen of oseltamivir for 5 days 72 h after intranasal (IN) infection with 10×MLD₅₀ of A/Puerto Rico/08/1934 (H1N1). Results of three independent trials (N=15 per group) are shown.

FIG. 3. Shows the HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCDR1, LCDR2 and LCDR3 sequences for the antibody designated H1H11729P.

FIG. 4. Shows the HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCDR1, LCDR2 and LCDR3 amino acid sequences for the antibody designated H1H11829N2.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

Definitions

The term “influenza hemagglutinin”, also called “influenza HA” is a trimeric glycoprotein found on the surface of influenza virions, which mediates viral attachment (via HA1 binding to α-2,3- and α-2,6-sialic acids) and entry (through conformational change) into host cells. The HA is comprised of two structural domains: a globular head domain containing the receptor binding site (subject to high frequency of antigenic mutations) and the stem region (more conserved among various strains of influenza virus). The influenza HA is synthesized as a precursor (HA0) that undergoes proteolytic processing to produce two subunits (HA1 and HA2) which associate with one another to form the stem/globular head structure. The viral HA is the most variable antigen on the virus (18 subtypes can be classified into two groups), but the stem (HA2) is highly conserved within each group.

The amino acid sequence of full-length Influenza HA is exemplified by the amino acid sequence of influenza isolate H1N1 A/California/04/2009 provided in GenBank as accession number FJ966082.1. The term “influenza-HA” also includes protein variants of influenza HA isolated from different influenza isolates, e.g., GQ149237.1, NC_002017, KM972981.1, etc. The term “influenza-HA” also includes recombinant influenza HA or a fragment thereof. The term also encompasses influenza HA or a fragment thereof coupled to, for example, histidine tag, mouse or human Fc, or a signal sequence.

The term “influenza infection”, as used herein, also characterized as “flu” refers to the severe acute respiratory illness caused by influenza virus. The term includes respiratory tract infection and the symptoms that include high fever, headache, general aches and pains, fatigue and weakness, in some instances extreme exhaustion, stuffy nose, sneezing, sore throat, chest discomfort, cough, shortness of breath, bronchitis, pneumonia and death in severe cases.

The term “antibody”, as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., “full antibody molecules”), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region (“HCVR” or “V_(H)”) and a heavy chain constant region (comprised of domains C_(H)1, C_(H)2 and C_(H)3). Each light chain is comprised of a light chain variable region (“LCVR or “V_(L)”) and a light chain constant region (CL). The V_(H) and V_(L) regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_(H) and V_(L) is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens, based on published crystal structures, and concluded that only about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previous studies (for example residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

The fully human anti-influenza-HA monoclonal antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V_(H) and/or V_(L) domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired properties such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

The present invention also includes fully human anti-influenza-HA monoclonal antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-influenza-HA antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody”, as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal, or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

The term “recombinant”, as used herein, refers to antibodies or antigen-binding fragments thereof of the invention created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

The term “specifically binds,” or “binds specifically to”, or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10⁻⁸ M or less (e.g., a smaller K_(D) denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by real-time, label free bio-layer interferometry assay on an Octet® HTX biosensor, which bind specifically to influenza-HA. Moreover, multi-specific antibodies that bind to one domain in influenza-HA and one or more additional antigens or a bi-specific that binds to two different regions of influenza-HA are nonetheless considered antibodies that “specifically bind”, as used herein.

The term “high affinity” antibody refers to those mAbs having a binding affinity to influenza-HA, expressed as K_(D), of at least 10⁻⁸ M; preferably 10⁻⁹ M; more preferably 10⁻¹⁰ M, even more preferably 10⁻¹¹ M, even more preferably 10⁻¹² M, as measured by real-time, label free bio-layer interferometry assay, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by solution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody that dissociates from influenza-HA, with a rate constant of 1×10⁻³ s⁻¹ or less, preferably 1×10⁻⁴ s⁻¹ or less, as determined by real-time, label free bio-layer interferometry assay, e.g., an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™.

The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms “antigen-binding fragment” of an antibody, or “antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to Influenza HA.

In specific embodiments, antibody or antibody fragments of the invention may be conjugated to a moiety such a ligand or a therapeutic moiety (“immunoconjugate”), such as an anti-viral drug, a second anti-influenza antibody, or any other therapeutic moiety useful for treating an infection caused by influenza-HA.

An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds influenza-HA, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than influenza-HA.

A “blocking antibody” or a “neutralizing antibody”, as used herein (or an “antibody that neutralizes influenza-HA activity” or “antagonist antibody”), is intended to refer to an antibody whose binding to influenza-HA results in inhibition of at least one biological activity of influenza-HA. For example, an antibody of the invention may prevent or block influenza attachment to, or entry into a host cell. In addition, a “neutralizing antibody” is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms “neutralizing antibody” and “an antibody that neutralizes” or “antibodies that neutralize” are used interchangeably herein. These antibodies can be used, alone or in combination, as prophylactic or therapeutic agents with other anti-viral agents upon appropriate formulation, or in association with active vaccination, or as a diagnostic tool.

The term “surface plasmon resonance”, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J

Bio-layer interferometry is a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time (Abdiche, Y. N., et al. Analytical Biochemistry, (2008), 377(2), 209-217). In certain embodiments of the invention, a “real-time bio-layer interferometer based biosensor (Octet HTX assay)” was used to assess the binding characteristics of certain of the anti-influenza HA antibodies.

The term “K_(D)”, as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term “epitope” refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.

The term “cross-competes”, as used herein, means an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice-versa. In certain embodiments, the first antibody and second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different, but overlapping epitopes such that binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Cross-competition between antibodies may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. Cross-competition between two antibodies may be expressed as the binding of the second antibody that is less than the background signal due to self-self binding (wherein first and second antibodies is the same antibody). Cross-competition between 2 antibodies may be expressed, for example, as % binding of the second antibody that is less than the baseline self-self background binding (wherein first and second antibodies is the same antibody).

The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated by reference.

By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term “subject” refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention and/or treatment of a disease or disorder such as viral infection. The subject may have an influenza infection or is predisposed to developing an influenza virus infection. Subjects “predisposed to developing an influenza virus infection”, or subjects “who may be at elevated risk for contracting an influenza virus infection”, are those subjects with compromised immune systems because of autoimmune disease, those persons receiving immunosuppressive therapy (for example, following organ transplant), those persons afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), certain forms of anemia that deplete or destroy white blood cells, those persons receiving radiation or chemotherapy, or those persons afflicted with an inflammatory disorder. Additionally, subject of extreme young or old age are at increased risk. Any person who comes into physical contact or close physical proximity with an infected individual has an increased risk of developing an Influenza virus infection. Moreover, a subject is at risk of contracting an influenza infection due to proximity to an outbreak of the disease, e.g. subject resides in a densely-populated city or in close proximity to subjects having confirmed or suspected infections of Influenza virus, or choice of employment, e.g. hospital worker, pharmaceutical researcher, traveler to infected area, or frequent flier.

As used herein, the terms “treat”, “treating”, or “treatment” refer to the reduction or amelioration of the severity of at least one symptom or indication of influenza infection due to the administration of a therapeutic agent such as an antibody of the present invention to a subject in need thereof. The terms include inhibition of progression of disease or of worsening of infection. The terms also include positive prognosis of disease, i.e., the subject may be free of infection or may have reduced or no viral titers upon administration of a therapeutic agent such as an antibody of the present invention. The therapeutic agent may be administered at a therapeutic dose to the subject.

The terms “prevent”, “preventing” or “prevention” refer to inhibition of manifestation of influenza infection or any symptoms or indications of influenza infection upon administration of an antibody of the present invention. The term includes prevention of spread of infection in a subject exposed to the virus or at risk of having influenza infection.

As used herein a “protective effect” may be demonstrated by any standard procedure known in the art to determine whether an agent such as an anti-viral agent, or an antibody such as an anti-influenza-HA antibody of the invention can demonstrate any one or more of the following: e.g. an increase in survival after exposure to an infectious agent, a decrease in viral load, or amelioration of at least one symptom associated with the infectious agent.

As used herein, the term “anti-viral drug” refers to any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. The term “anti-viral drug” includes, but is not limited to TAMIFLU® (Oseltamivir), RELENZA® (Zanamivir), ribavirin, or interferon-alpha2b. In the present invention, the infection to be treated is caused by an influenza virus.

General Description

Influenza is an infectious disease caused by RNA viruses of the family Orthomyxoviridae (the influenza viruses). Influenza viruses are classified based on core protein into three genera A, B and C that are further divided into subtypes determined by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA). Influenza A viruses infect a range of mammalian and avian species, whereas type B and C infections are largely restricted to humans. Only types A and B cause human disease of any concern.

High mutation rates and frequent genetic reassortments of the influenza viruses contribute to great variability of the HA and NA antigens. Minor point mutations causing small changes (“antigenic drift”) occur relatively often. Antigenic drift enables the virus to evade immune recognition, resulting in repeated influenza outbreaks during interpandemic years. Major changes in the HA antigen (“antigenic shift”) are caused by reassortment of genetic material from different influenza A subtypes. Antigenic shifts resulting in new pandemic strains are rare events, occurring through reassortment between animal and human subtypes, for example in co-infected pigs.

The neutralizing antibody response to Influenza A virus is typically specific for a given viral subtype. There are 18 influenza A subtypes defined by their hemagglutinin (“HA”) proteins. The 18 HAs, H1-H18, can be classified into two groups. Group 1 consists of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18 subtypes, and group 2 includes H3, H4, H7, H10, H14 and H15 subtypes. For these reasons it would be highly desirable to have a vaccine that induces broadly neutralizing antibodies capable of neutralizing all influenza A virus subtypes as well as their yearly variants. In addition broadly neutralizing heterosubtypic antibodies could be administered as medicaments for prevention or therapy of influenza A infection.

HA is synthesized as a homo-trimeric precursor polypeptide HA0. Each monomer can be independently cleaved post-translationally to form two polypeptides, HA1 and HA2, linked by a single disulphide bond. The larger N-terminal fragment (HAL 320-330 amino acids) forms a membrane-distal globular domain that contains the receptor-binding site and most determinants recognized by virus-neutralizing antibodies. The HA1 polypeptide of HA is responsible for the attachment of virus to the cell surface. The smaller C-terminal portion (HA2, approximately 180 amino acids) forms a stem-like structure that anchors the globular domain to the cellular or viral membrane. The HA2 polypeptide mediates the fusion of viral and cell membranes in endosomes, allowing the release of the ribonucleoprotein complex into the cytoplasm.

There has only been limited success in identifying antibodies that neutralize more than one subtype of influenza A virus. Further, the breath of neutralization of antibodies identified thus far is narrow and their potency is low. Okuno et al, immunized mice with influenza virus A/Okuda/57 (H2N2) and isolated a monoclonal antibody (C179) that binds to a conserved conformational epitope in HA2 and neutralizes the Group 1 H2, H1 and H5 subtype influenza A viruses in vitro and in vivo in animal models ((Okuno et al., J. Virol. 67:2552-8, 1993).

Despite decades of research, there are no marketed antibodies that broadly neutralize or inhibit influenza A virus infection or attenuate disease caused by influenza A virus. Therefore, there is a need to identify new antibodies that neutralize multiple subtypes of influenza A virus and can be used as medicaments for prevention or therapy of influenza A infection.

Passive immunotherapy for prophylaxis or treatment of infectious diseases has been used for more than a century, usually in the form of convalescent human sera that contains high titers of neutralizing antibodies (Good et al. 1991; Cancer 68: 1415-1421). Today, multiple purified monoclonal antibodies are currently in preclinical and clinical development for use as anti-microbials (Marasco et al. 2007; Nature Biotechnology 25: 1421-1434).

The inventors have described herein fully human antibodies and antigen-binding fragments thereof that specifically bind to influenza hemagglutinin and modulate the interaction of influenza virus with host cells. The anti-influenza HA antibodies may bind to the influenza virus HA with high affinity. In certain embodiments, the antibodies of the present invention are blocking antibodies wherein the antibodies may bind to influenza HA and block the attachment to and/or entry of the virus into host cells. In some embodiments, the blocking antibodies of the invention may block the binding of influenza virus to cells and as such may inhibit or neutralize viral infectivity of host cells. In some embodiments, the blocking antibodies may be useful for treating a subject suffering from an influenza virus infection. The antibodies when administered to a subject in need thereof may reduce the infection by a virus such as influenza in the subject. They may be used to decrease viral loads in a subject. They may be used alone or as adjunct therapy with other therapeutic moieties or modalities known in the art for treating a viral infection. In certain embodiments, these antibodies may bind to an epitope in the stem region of the viral HA. Furthermore, the identified antibodies can be used prophylactically (before infection) to protect a mammal from infection, or can be used therapeutically (after infection is established) to ameliorate a previously established infection, or to ameliorate at least one symptom associated with the infection.

The full-length amino acid sequence of an exemplary Influenza HA is shown in GenBank as accession number HC483324.1 (See SEQ ID NO: 62 in PCT publication WO2010/027818).

In certain embodiments, the antibodies of the invention are obtained from mice immunized with a primary immunogen, such as a full length influenza HA or with a recombinant form of influenza HA or fragments thereof followed by immunization with a secondary immunogen, or with an immunogenically active fragment of influenza HA. In certain embodiments, the antibodies are obtained from mice immunized with an influenza vaccine composition followed by booster immunization with one or more recombinantly produced HA peptides.

The immunogen may be a biologically active and/or immunogenic fragment of influenza HA or DNA encoding the active fragment thereof. The fragment may be derived from the stem region of the HA protein. (See Sui et. al., Nature Struct. and Mol. Biol. Published online 22 Feb. 2009; Pages 1-9).

The peptides may be modified to include addition or substitution of certain residues for tagging or for purposes of conjugation to carrier molecules, such as, KLH. For example, a cysteine may be added at either the N terminal or C terminal end of a peptide, or a linker sequence may be added to prepare the peptide for conjugation to, for example, KLH for immunization.

Certain anti-influenza-HA antibodies of the present invention are able to bind to and neutralize the activity of influenza-HA, as determined by in vitro or in vivo assays. The ability of the antibodies of the invention to bind to and neutralize the activity of influenza-HA and thus the attachment and/or entry of the virus into a host cell followed by the ensuing viral infection, may be measured using any standard method known to those skilled in the art, including binding assays, or activity assays, as described herein.

Non-limiting, exemplary in vitro assays for measuring binding activity are illustrated in Example 3, herein. In Example 3, the binding affinity and dissociation constants of anti-influenza-HA antibodies for influenza-HA were determined by real-time bio-layer interferometer based biosensor (Octet HTX assay). In Examples 4 and 5, neutralization assays were used to determine infectivity of diverse group 1 strains of influenza virus. In Example 6, certain antibodies were shown to mediate complement dependent cytotoxicity (CDC) of virus-infected cells in vitro. Examples 7 and 10 demonstrate that certain antibodies of the invention are capable of neutralizing an influenza A infection in vivo when administered either prophylactically or therapeutically.

The antibodies specific for influenza-HA may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye or a MRI-detectable label. In certain embodiments, such labeled antibodies may be used in diagnostic assays including imaging assays.

Antigen-Binding Fragments of Antibodies

Unless specifically indicated otherwise, the term “antibody,” as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., “full antibody molecules”) as well as antigen-binding fragments thereof. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms “antigen-binding fragment” of an antibody, or “antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to Influenza HA. An antibody fragment may include a Fab fragment, a F(ab′)₂ fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term “antigen-binding fragment” refers to a polypeptide fragment of a multi-specific antigen-binding molecule. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V_(H) domain associated with a V_(L) domain, the V_(H) and V_(L) domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) or V_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V_(H)-C_(H)1; (ii) V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (V) V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; V_(H)—CL; V_(L)—C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi) V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii) V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V_(H) or V_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may be mono-specific or multi-specific (e.g., bi-specific). A multi-specific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antibody format, including the exemplary bi-specific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to Influenza-HA. An immunogen comprising any one of the following can be used to generate antibodies to Influenza HA. In certain embodiments, the antibodies of the invention are obtained from mice immunized with a full length, native influenza HA (See, for example, GenBank accession number FJ966082.1), or with a live attenuated or inactivated virus, or with DNA encoding the protein or fragment thereof. Alternatively, the influenza-HA protein or a fragment thereof may be produced using standard biochemical techniques and modified and used as immunogen. In one embodiment, the immunogen is a recombinantly produced influenza-HA protein or fragment thereof. In certain embodiments of the invention, the immunogen may be an influenza virus vaccine. In certain embodiments, one or more booster injections may be administered. In certain embodiments, the booster injections may comprise one or more influenza virus strains, or hemagglutinins derived from these strains, eg., See Protein Sciences H1 A/New Caledonia/20/1999, H5 A/Indonesia/05/2005, H3 A/Victoria/361/2011, H7 A/Netherlands/219/2003, or H9 A/Hong Kong/1073/1988. In certain embodiments, the booster injections may contain a 1:1 mixture of the influenza strains, or a 1:1 mixture of the hemagglutinins derived from the strains. In certain embodiments, the immunogen may be a recombinant Influenza HA peptide expressed in E. coli or in any other eukaryotic or mammalian cells such as Chinese hamster ovary (CHO) cells or influenza virus itself.

Using VELOCIMMUNE® technology (see, for example, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to influenza-HA are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

Bioequivalents

The anti-influenza-HA antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind Influenza HA. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an antibody or antibody fragment that is essentially bioequivalent to an antibody or antibody fragment of the invention.

Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

Bioequivalent variants of the antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, e.g., mutations that eliminate or remove glycosylation.

Anti-Influenza-HA Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-influenza-HA antibodies are provided comprising an Fc domain comprising one or more mutations, which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present invention includes anti-influenza-HA antibodies comprising a mutation in the C_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modification comprises a 265A (e.g., D265A) and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-influenza-HA antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present invention.

The present invention also includes anti-influenza-HA antibodies comprising a chimeric heavy chain constant (C_(H)) region, wherein the chimeric C_(H) region comprises segments derived from the C_(H) regions of more than one immunoglobulin isotype. For example, the antibodies of the invention may comprise a chimeric C_(H) region comprising part or all of a C_(H)2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule, combined with part or all of a C_(H)3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibodies of the invention comprise a chimeric C_(H) region having a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. An antibody comprising a chimeric C_(H) region as described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., U.S. Provisional Appl. No. 61/759,578, filed Feb. 1, 2013, the disclosure of which is hereby incorporated by reference in its entirety).

Biological Characteristics of the Antibodies

In general, the antibodies of the present invention function by binding to Influenza HA. For example, the present invention includes antibodies and antigen-binding fragments of antibodies that bind Influenza HA (e.g., at 25° C. or at 37° C.) with a K_(D) of less than 10 nM, as measured by real-time bio-layer interferometer based biosensor (Octet HTX assay), or by surface plasmon resonance. In certain embodiments, the antibodies or antigen-binding fragments thereof bind influenza-HA with a K_(D) of less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, less than 250 pM, or less than 100 pM, as measured by surface plasmon resonance, e.g., using the assay format as described herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding fragments thereof that bind Influenza HA with a dissociative half-life (t½) of greater than about 100 minutes as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind Influenza HA with a t½ of greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, or greater than about 1000 minutes as measured by surface plasmon resonance at 25° C., e.g., using an assay format as defined herein (e.g., mAb-capture or antigen-capture format), or a substantially similar assay. In one embodiment, the antibodies and antigen-binding fragments of the invention bind Influenza HA with a dissociative half-life (t½) of greater than 300 minutes. In one embodiment, an antibody of the invention provides for about a 1.5 to 2-fold increase in dissociative half life as compared to a comparator antibody designated Control I mAb, when tested in monkeys and mice.

The present invention also includes antibodies or antigen-binding fragments thereof that neutralize the infectivity of influenza virus for its host cells. In some embodiments, the antibodies exhibit a neutralization potency against various representative group 1 influenza viruses (H1N1 A/Puerto Rico/08/1934; H5N1 A/Vietnam/1203/2004; H1N1 A/California/07/2009; H1N1 A/Wisconsin/1933; H1N1 A/Brisbane/59/1997, H9N2 A/Hong Kong/33982/2009, H13N6 a/gull/Maryland/704/1977 and H16N3 A/shorebird/Delaware/172/2006 with an IC₅₀ ranging from about 1.6 nM to about 130 nM in a microneutralization assay, e.g., as shown in Examples 4 and 5, or a substantially similar assay. In one embodiment, the antibodies or antigen-binding fragments thereof that neutralize the infectivity of influenza virus for its host cells do so with an IC₅₀ of less than 130 nM.

The present invention also includes antibodies or antigen-binding fragments thereof that mediate complement-dependent cytotoxicity of infected cells, with an EC₅₀ ranging from about 20 nM to about 66 nM (see example 6). In one embodiment, the antibodies or antigen-binding fragments thereof mediate complement-dependent cytotoxicity of infected cells, with an EC₅₀ less than 66 nM.

The present invention also includes anti-influenza-A HA antibodies that demonstrate an increase in protection, or potent neutralization of influenza A infection in vivo. Certain antibodies show potent neutralization when administered either prophylactically (prior to infection) or therapeutically (after infection; see example 7). In certain embodiments, some of the antibodies (H1H11729P and H1H11829N2) demonstrated 100% survival of mice when administered prophylactically as a single dose of 1 mg/kg. Certain antibodies demonstrated significant survival of mice when administered prophylactically at doses as low as 0.5 mg/kg (100% survival using the antibody designated as H1H11729P), at 0.1 mg/kg of H1H11729P (40% survival), or at 0.05 mg/kg of H1H11829N2 (20% survival). Significant survival was also observed when certain exemplary antibodies (H1H11829N2 and H1H11729P) were administered after infection at doses of 15 or 30 mg/kg. In one embodiment, an antibody of the invention demonstrates an additive protective effect in influenza-infected mammals when combined with an anti-viral drug, oseltamivir.

In one embodiment, the invention provides an isolated recombinant antibody or antigen-binding fragment thereof that binds specifically to Influenza HA, wherein the antibody or fragment thereof exhibits two or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to influenza HA with a dissociation constant (K_(D)) of less than 10⁻⁹M, as measured in a surface plasmon resonance assay; (c) demonstrates a dissociative half-life (t½) ranging from about 370 minutes to greater than 1000 minutes; (d) demonstrates neutralization of group 1 influenza A viruses selected from H1N1, H5N1, H9N2, H13N6 and H16N3, with an IC₅₀ ranging from about 1.6 nM to about 130 nM; (e) demonstrates complement mediated lysis of influenza virus infected cells with an EC₅₀ of about 20 nM to about 66 nM; or (f) demonstrates protection, as measured by increased survival in an animal model of influenza virus infection when administered either before or after virus challenge.

The antibodies of the present invention may possess two or more of the aforementioned biological characteristics, or any combinations thereof. Other biological characteristics of the antibodies of the present invention will be evident to a person of ordinary skill in the art from a review of the present disclosure including the working Examples herein.

Epitope Mapping and Related Technologies

The present invention includes anti-influenza-HA virus antibodies, which interact with one or more amino acids found within one or more domains of the influenza HA molecule. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within the influenza HA molecule (e.g. a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the influenza-HA molecule (e.g. a conformational epitope).

Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody “interacts with one or more amino acids” within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

The term “epitope” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the invention into groups of antibodies binding different epitopes.

In certain embodiments, the influenza virus-HA antibodies or antigen-binding fragments thereof bind an epitope within any one or more of the regions exemplified in influenza HA, either in natural form, or recombinantly produced, or to a fragment thereof.

The present invention includes anti-influenza-HA antibodies that bind to the same epitope, or a portion of the epitope. Likewise, the present invention also includes anti-influenza-HA antibodies that compete for binding to Influenza HA or a fragment thereof with any of the specific exemplary antibodies described herein. For example, the present invention includes anti-influenza-HA antibodies that cross-compete for binding to influenza HA with one or more antibodies obtained from those antibodies described in Tables 1 and 12.

One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-influenza-HA antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-influenza-HA antibody of the invention, the reference antibody is allowed to bind to an Influenza HA or peptide under saturating conditions. Next, the ability of a test antibody to bind to the Influenza HA molecule is assessed. If the test antibody is able to bind to influenza virus HA following saturation binding with the reference anti-influenza-HA antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-influenza-HA antibody. On the other hand, if the test antibody is not able to bind to the Influenza HA following saturation binding with the reference anti-influenza-HA antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-influenza-HA antibody of the invention.

To determine if an antibody competes for binding with a reference anti-influenza-HA antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a Influenza HA under saturating conditions followed by assessment of binding of the test antibody to the influenza-HA molecule. In a second orientation, the test antibody is allowed to bind to an influenza-HA molecule under saturating conditions followed by assessment of binding of the reference antibody to the influenza-HA molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the influenza-HA molecule, then it is concluded that the test antibody and the reference antibody compete for binding to influenza-HA. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.

Immunoconjugates

The invention encompasses a human anti-influenza-HA monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), such as a toxoid or an anti-viral drug to treat influenza virus infection. As used herein, the term “immunoconjugate” refers to an antibody, which is chemically or biologically linked to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein or a therapeutic agent. The antibody may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any location along the molecule so long as it is able to bind its target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second different antibody to Influenza-HA. In certain embodiments, the antibody may be conjugated to an agent specific for a virally infected cell. The type of therapeutic moiety that may be conjugated to the anti-influenza-HA antibody and will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see for example, WO 05/103081.

Multi-Specific Antibodies

The antibodies of the present invention may be mono-specific, bi-specific, or multi-specific. Multi-specific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

Any of the multi-specific antigen-binding molecules of the invention, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology), as will be known to a person of ordinary skill in the art.

In some embodiments, influenza-HA-specific antibodies are generated in a bi-specific format (a “bi-specific”) in which variable regions binding to distinct domains of Influenza HA are linked together to confer dual-domain specificity within a single binding molecule. Appropriately designed bi-specifics may enhance overall influenza-HA-protein inhibitory efficacy through increasing both specificity and binding avidity. Variable regions with specificity for individual domains, (e.g., segments of the N-terminal domain), or that can bind to different regions within one domain, are paired on a structural scaffold that allows each region to bind simultaneously to the separate epitopes, or to different regions within one domain. In one example for a bi-specific, heavy chain variable regions (V_(H)) from a binder with specificity for one domain are recombined with light chain variable regions (V_(L)) from a series of binders with specificity for a second domain to identify non-cognate V_(L) partners that can be paired with an original V_(H) without disrupting the original specificity for that V_(H). In this way, a single V_(L) segment (e.g., V_(L)1) can be combined with two different V_(H) domains (e.g., V_(H)1 and V_(H)2) to generate a bi-specific comprised of two binding “arms” (V_(H)1-V_(L)1 and V_(H)2-V_(L)1). Use of a single V_(L) segment reduces the complexity of the system and thereby simplifies and increases efficiency in cloning, expression, and purification processes used to generate the bi-specific (See, for example, U.S. Ser. No. 13/022,759 and US2010/0331527).

Alternatively, antibodies that bind more than one domain and a second target, such as, but not limited to, for example, a second different anti-influenza-HA antibody, may be prepared in a bi-specific format using techniques described herein, or other techniques known to those skilled in the art. Antibody variable regions binding to distinct regions may be linked together with variable regions that bind to relevant sites on, for example, the influenza virus, to confer dual-antigen specificity within a single binding molecule. Appropriately designed bi-specifics of this nature serve a dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for outside the extracellular domain and are paired on a structural scaffold that allows each variable region to bind to the separate antigens.

An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first and second Ig C_(H)3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi-specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C_(H)3 domain binds Protein A and the second Ig C_(H)3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C_(H)3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

Other exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab^(e) bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-influenza-HA antibodies or antigen-binding fragments thereof of the present invention. Therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When an antibody of the present invention is used for treating a disease or disorder in an adult patient, or for preventing such a disease, it is advantageous to administer the antibody of the present invention normally at a single dose of about 0.1 to about 60 mg/kg body weight, more preferably about 5 to about 60, about 10 to about 50, or about 20 to about 50 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention can be administered as an initial dose of at least about 0.1 mg to about 5000 mg, about 1 to about 2000 mg, about 5 to about 1000 mg, or about 10 to about 500 mg, to about 100 mg, or to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249:1527-1533).

The use of nanoparticles to deliver the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles may be used both for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo, M., et al. 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporated herein by reference. Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target virally infected cells. Nanoparticles for drug delivery have also been described in, for example, U.S. Pat. No. 8,257,740, or U.S. Pat. No. 8,246,995, each incorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.

The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 5000 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the antibody is contained in about 5 to about 500 mg and in about 10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the present invention are useful for the treatment, and/or prevention of a disease or disorder or condition associated with influenza virus infection and/or for ameliorating at least one symptom associated with such disease, disorder or condition.

In certain embodiments, the antibodies of the invention are useful to treat subjects suffering from the severe and acute respiratory infection caused by influenza virus. In some embodiments, the antibodies of the invention are useful in decreasing viral titers or reducing viral load in the host. In one embodiment, an antibody or antigen-binding fragment thereof the invention may be administered at a therapeutic dose to a patient with influenza virus infection.

One or more antibodies of the present invention may be administered to relieve or prevent or decrease the severity of one or more of the symptoms or conditions of the disease or disorder. The antibodies may be used to ameliorate or reduce the severity of at least one symptom of influenza virus infection including, but not limited to fever, cough, sore throat, headache, body aches, fatigue, extreme exhaustion, shortness of breath, bronchitis, pneumonia, and death.

It is also contemplated herein to use one or more antibodies of the present invention prophylactically to subjects at risk for developing an influenza virus infection such as immunocompromised individuals, elderly adults (more than 65 years of age), children younger than 2 years of age, healthcare workers, family members in close proximity to a patient suffering from an influenza virus infection, and patients with a medical history (e.g., increased risk of pulmonary infection, heart disease or diabetes).

In a further embodiment of the invention the present antibodies are used for the preparation of a pharmaceutical composition for treating patients suffering from an influenza virus infection. In another embodiment of the invention, the present antibodies are used as adjunct therapy with any other agent or any other therapy known to those skilled in the art useful for treating or ameliorating an influenza virus infection.

Combination Therapies

Combination therapies may include an anti-influenza-HA antibody of the invention and any additional therapeutic agent that may be advantageously combined with an antibody of the invention, or with a biologically active fragment of an antibody of the invention. The antibodies of the present invention may be combined synergistically with one or more drugs or agents (e.g. anti-viral agents) used to treat influenza virus.

For example, exemplary anti-viral agents include, e.g., vaccines, neuraminidase inhibitors or nucleoside analogs. Other exemplary anti-viral agents that may be used in combination with an antibody of the invention can include, e.g., zidovudine, gangcyclovir, vidarabine, idoxuridine, trifluridine, foscarnet, acyclovir, ribavirin, amantadine, remantidine, saquinavir, indinavir, ritonavir, alpha-interferons and other interferons, a neuraminidase inhibitor (e.g., zanamivir (RELENZA®), oseltamivir (TAMIFLU®) laninamivir, peramivir), or rimantadine.

Other exemplary anti-viral drugs include, but are not limited to, a HA inhibitor, a sialic acid inhibitor and an M2 ion channel inhibitor. In one embodiment, the M2 ion channel inhibitor is amantadine or rimantadine.

In some embodiments, the antibodies of the invention may be combined with a second therapeutic agent to reduce the viral load in a patient with an influenza virus infection, or to ameliorate one or more symptoms of the infection.

The antibodies of the present invention may be used in combination with an anti-inflammatory drug (e.g., corticosteroids, and non-steroidal anti-inflammatory drugs), a decongestant, an anti-histamine, an anti-infective drug, a different antibody to Influenza virus, an anti-viral drug, a vaccine for influenza virus, such as FLUMIST® or FLUVIRIN®, a dietary supplement such as anti-oxidants or any other palliative therapy to treat an influenza virus infection.

In certain embodiments, the second therapeutic agent is another antibody to influenza. In certain embodiments, the second therapeutic agent is another antibody to influenza hemagglutinin. In certain embodiments, the second therapeutic agent is another antibody to a different influenza protein, such as the neuraminidase, or the tetrameric ectodomain of matrix protein 2 (M2e protein). In certain embodiments, the second therapeutic agent is an antibody to a different protein such as the host transmembrane protease, serine 2 (TMPRSS2). The second antibody may be specific for one or more different influenza virus proteins from different subtypes or strains of the virus. It is contemplated herein to use a combination (“cocktail”) of antibodies with broad neutralization or inhibitory activity against influenza virus. In some embodiments, non-competing antibodies may be combined and administered to a subject in need thereof, to reduce the ability of influenza virus to escape due to rapid mutation as a result of selection pressure. In some embodiments, the antibodies comprising the combination bind to distinct non-overlapping epitopes on the HA protein. The antibodies comprising the combination may block the virus attachment and/or entry into and/or fusion with host cells. The antibodies may interact with a hemagglutinin selected from any one or more of the Group 1 influenza A subtypes including H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 or H18 and when used alone, or in combination with any one or more of the agents noted above, may neutralize any one or more of the Group 1 influenza subtypes including, but not limited to the following: H1N1, H5N1, H9N2, H13N6 and H16N3.

It is also contemplated herein to use a combination of anti-influenza-HA antibodies of the present invention, wherein the combination comprises one or more antibodies that do not cross-compete; In some embodiments, the combination includes a first antibody with broad neutralization activity with a second antibody with activity against a narrow spectrum of isolates and that does not cross-compete with the first antibody.

As used herein, the term “in combination with” means that additional therapeutically active component(s) may be administered prior to, concurrent with, or after the administration of the anti-influenza-HA antibody of the present invention. The term “in combination with” also includes sequential or concomitant administration of an anti-influenza-HA antibody and a second therapeutic agent.

The additional therapeutically active component(s) may be administered to a subject prior to administration of an anti-influenza-HA antibody of the present invention. For example, a first component may be deemed to be administered “prior to” a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject after administration of an anti-influenza-HA antibody of the present invention. For example, a first component may be deemed to be administered “after” a second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In yet other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with administration of an anti-influenza-HA antibody of the present invention. “Concurrent” administration, for purposes of the present invention, includes, e.g., administration of an anti-influenza-HA antibody and an additional therapeutically active component to a subject in a single dosage form, or in separate dosage forms administered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-influenza-HA antibody and the additional therapeutically active component may be administered intravenously, etc.); alternatively, each dosage form may be administered via a different route (e.g., the anti-influenza-HA antibody may be administered intravenously, and the additional therapeutically active component may be administered orally). In any event, administering the components in a single dosage from, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered “concurrent administration,” for purposes of the present disclosure. For purposes of the present disclosure, administration of an anti-influenza-HA antibody “prior to”, “concurrent with,” or “after” (as those terms are defined herein above) administration of an additional therapeutically active component is considered administration of an anti-influenza-HA antibody “in combination with” an additional therapeutically active component.

The present invention includes pharmaceutical compositions in which an anti-influenza-HA antibody of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.

Administration Regimens

According to certain embodiments, a single dose of an anti-influenza-HA antibody of the invention (or a pharmaceutical composition comprising a combination of an anti-influenza-HA antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject in need thereof. According to certain embodiments of the present invention, multiple doses of an anti-influenza-HA antibody (or a pharmaceutical composition comprising a combination of an anti-influenza-HA antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an anti-influenza-HA antibody of the invention. As used herein, “sequentially administering” means that each dose of anti-influenza-HA antibody is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of an anti-influenza-HA antibody, followed by one or more secondary doses of the anti-influenza-HA antibody, and optionally followed by one or more tertiary doses of the anti-influenza-HA antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the anti-influenza-HA antibody of the invention. Thus, the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of anti-influenza-HA antibody, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of anti-influenza-HA antibody contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).

In certain exemplary embodiments of the present invention, each secondary and/or tertiary dose is administered 1 to 48 hours (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½.19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of anti-influenza-HA antibody, which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an anti-influenza-HA antibody. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

In certain embodiments of the invention, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

Diagnostic Uses of the Antibodies

The anti-influenza-HA antibodies of the present invention may be used to detect and/or measure influenza-HA in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present invention in assays to detect a disease or disorder such as viral infection. Exemplary diagnostic assays for influenza-HA may comprise, e.g., contacting a sample, obtained from a patient, with an anti-influenza-HA antibody of the invention, wherein the anti-influenza-HA antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate influenza-HA from patient samples. Alternatively, an unlabeled anti-influenza-HA antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure influenza-HA in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in influenza-HA diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of either influenza HA, or fragments thereof, under normal or pathological conditions. Generally, levels of influenza HA in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease associated with influenza will be measured to initially establish a baseline, or standard, level of influenza-HA. This baseline level of influenza-HA can then be compared against the levels of influenza-HA measured in samples obtained from individuals suspected of having a influenza-HA-associated condition, or symptoms associated with such condition.

The antibodies specific for influenza HA may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25° C., and pressure is at or near atmospheric.

Example 1: Generation of Human Antibodies to Influenza HA

Human antibodies to influenza HA were generated in a VELOCIMMUNE® mouse comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions. The mice were immunized with an influenza vaccine composition, followed by a booster dose comprising a mixture of five different recombinant hemagglutinin proteins, at a 1:1 ratio of each. The five recombinant hemagglutinin proteins included in the booster were hemagglutinins from H1 A/New Caledonia/20/1999, H5 A/Indonesia/05/2005, H3 A/Victoria/361/2011, H7 A/Netherlands/219/2003 and H9 A/Hong Kong/1073/1988 (Protein Sciences, Catalog Number 3006). The antibody immune response was monitored by an influenza A HA specific immunoassay. When a desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce influenza HA-specific antibodies. Using this technique, and the various immunogens described above, several chimeric antibodies (i.e., antibodies possessing human variable domains and mouse constant domains) were obtained; exemplary antibodies generated in this manner were designated as H1H11820N, H1H11829N, H1H11829N2, H2M11830N, H1H11830N2, H1H11903N and H1H14571N.

Anti-influenza HA antibodies were also isolated directly from antigen-positive mouse B cells without fusion to myeloma cells, as described in U.S. Pat. No. 7,582,298, herein specifically incorporated by reference in its entirety. Using this method, several fully human anti-influenza HA antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner were designated as H1H11723P, H1H11729P, H1H11704P, H1H11711P, H1H11714P, H1H11717P, H1H11724P, H1H11727P, H1H11730P, H1H11731P2, H1H11734P2, H1H11736P2, H1H11742P2, H1H11744P2, H1H11745P2, H1H11747P2, H1H11748P2.

The biological properties of the exemplary antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.

Example 2: Heavy and Light Chain Variable Region Amino Acid and Nucleotide Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-influenza HA antibodies of the invention. The corresponding nucleic acid sequence identifiers are set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H11723P 2 4 6 8 10 12 14 16 H1H11729P 18 20 22 24 26 28 30 32 H1H11820N 34 36 38 40 42 44 46 48 H1H11829N 50 52 54 56 58 60 62 64 H1H11829N2 50 52 54 56 66 68 70 72 H2aM11829N 50 52 54 56 58 60 62 64 H2M11830N 74 76 78 80 82 84 86 88 H1H11830N2 74 76 78 80 66 68 70 72 H1H11903N 90 92 94 96 98 100 102 104 H1H14571N 106 108 110 112 114 116 118 120 H2a14571N 106 108 110 112 114 116 118 120 H1H11704P 122 124 126 128 130 132 134 136 H1H11711P 138 140 142 144 146 148 150 152 H1H11714P 154 156 158 160 162 164 166 168 H1H11717P 170 172 174 176 178 180 182 184 H1H11724P 186 188 190 192 194 196 198 200 H1H11727P 202 204 206 208 210 212 214 216 H1H11730P2 218 220 222 224 226 228 230 232 H1H11731P2 234 236 238 240 66 68 70 72 H1H11734P2 242 244 246 248 66 68 70 72 H1H11736P2 250 252 254 256 66 68 70 72 H1H11742P2 258 260 262 264 66 68 70 72 H1H11744P2 266 268 270 272 66 68 70 72 H1H11745P2 274 276 278 280 66 68 70 72 H1H11747P2 282 284 286 288 66 68 70 72 H1H11748P2 290 292 294 296 66 68 70 72

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H11723P 1 3 5 7 9 11 13 15 H1H11729P 17 19 21 23 25 27 29 31 H1H11820N 33 35 37 39 41 43 45 47 H1H11829N 49 51 53 55 57 59 61 63 H1H11829N2 49 51 53 55 65 67 69 71 H2aM11829N 49 51 53 55 57 59 61 63 H2M11830N 73 75 77 79 81 83 85 87 H1H11830N2 73 75 77 79 65 67 69 71 H1H11903N 89 91 93 95 97 99 101 103 H1H14571N 105 107 109 111 113 115 117 119 H2a14571N 105 107 109 111 113 115 117 119 H1H11704P 121 123 125 127 129 131 133 135 H1H11711P 137 139 141 143 145 147 149 151 H1H11714P 153 155 157 159 161 163 165 167 H1H11717P 169 171 173 175 177 179 181 183 H1H11724P 185 187 189 191 193 195 197 199 H1H11727P 201 203 205 207 209 211 213 215 H1H11730P 217 219 221 223 225 227 229 231 H1H11731P2 233 235 237 239 65 67 69 71 H1H11734P2 241 243 245 247 65 67 69 71 H1H11736P2 249 251 253 255 65 67 69 71 H1H11742P2 257 259 261 263 65 67 69 71 H1H11744P2 265 267 269 271 65 67 69 71 H1H11745P2 273 275 277 279 65 67 69 71 H1H11747P2 281 283 285 287 65 67 69 71 H1H11748P2 289 291 293 295 65 67 69 71

Antibodies are typically referred to herein according to the following nomenclature: Fc prefix (e.g. “H1H,” “H2M,” etc.), followed by a numerical identifier (e.g. “11723,” “11830,” etc., as shown in Table 1 or 2), followed by a “P,” “P2,” “N”, N2, or “B” suffix. Thus, according to this nomenclature, an antibody may be referred to herein as, e.g., “H1H11723P,” “H2M11830N,” etc. The H1H and H2M prefixes on the antibody designations used herein indicate the particular Fc region isotype of the antibody. For example, an “H1 M” antibody has a mouse IgG1 Fc, and an “H2M” antibody has a mouse IgG2 Fc (a or b isotype) (all variable regions are fully human as denoted by the first ‘H’ in the antibody designation). As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted to an antibody with a human IgG4, etc.), but in any event, the variable domains (including the CDRs)—which are indicated by the numerical identifiers shown in Tables 1, 2, 12 or 13—will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the Fc domain.

Antibody Comparators

Anti-influenza-HA antibody controls were included in some of the following Examples for comparative purposes. Isotype matched negative controls were also used in the Examples. One anti-influenza HA comparator antibody, designated herein as “Control I mAb”, is an anti-influenza HA antibody with heavy (HC) and light chain (LC) amino acid sequences as set forth in WO2008/028946 as SEQ ID NO: 65 (HC) and SEQ ID NO: 91 (LC) and also referred to as CR6261. As shown in WO2008/028946, this CR6261 antibody has heavy chain complementarity determining region (HCDR) amino acid sequences (HCDR 1, 2 and 3) as shown as SEQ ID NOs: 1.2 and 3, respectively and light chain complementarity determining region (LCDR) amino acid sequences (LCDR1.2 and 3) as shown in SEQ ID NOs: 13, 14 and 15, respectively.

Example 3: Octet Binding Affinities and Kinetic Constants of Monoclonal Anti-Influenza HA Antibodies

Equilibrium dissociation constants (K_(D) values) for Influenza hemagglutinin (HA) binding to selected purified anti-HA monoclonal antibodies were determined using a real-time bio-layer interferometer based biosensor (Octet HTX) assay. Octet biosensors coated with either anti-mouse Fc (AMC) or anti-human Fc (AHC) capture were used to capture anti-HA monoclonal antibodies, expressed with mouse Fc (AbPID prefix H1M, H2aM, H2bM) or human IgG1 Fc (AbPID prefix H1H) respectively. All the binding studies were performed in HBS-ET Octet kinetics buffer (10 mM HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant Tween-20, 1 mg/mL BSA) at 25° C. with plates shaking at a speed of 1000 rpm. Antibody captured Octet biosensors were submerged in wells containing different concentrations of diverse strains of HA (300 nM to 11.11 nM) for 7 minutes followed by dissociation of antibody bound HA protein in Octet kinetics buffer for 10 minutes. Biosensors were always washed in Octet kinetics buffer in between different steps. Kinetic association (k_(a)) and dissociation (k_(d)) rate constants were determined by processing and fitting the data to a 1:1 binding model using Scrubber 2.0c curve fitting software. Binding dissociation equilibrium constants (K_(D)) and dissociative half-lives (t½) were calculated from the kinetic rate constants as:

${K_{D} = \frac{ka}{kd}},{{{and}\mspace{14mu} t\frac{1}{2}\mspace{14mu}\left( \min \right)} = \frac{\ln(2)}{60*{kd}}}$

Binding kinetics parameters for different anti-HA monoclonal antibodies binding to varying Group-1 strains of HA are tabulated in Table 3. In addition, the binding parameters for the same antibodies were determined for binding to group 2 hemagglutinins, however, there was no binding to group 2 HA (data not shown).

TABLE 3 Binding Kinetics parameters of monoclonal antibodies binding to different HA strains of Group-1 at 25° C. Amount of 300 nM mAb HA mAb Injected Captured Bound k_(a) k_(d) K_(D) t½ Captured Analyte (nm) (nm) (1/Ms) (1/s) (M) (min) H2aM11829N A/California/ 0.53 ± 0.01 0.62 1.20E+05  1.00E−05* 8.35E−11 1155* 07/2009 (H1N1) A/Puerto 0.53 ± 0.01 0.59 1.09E+05 1.16E−05 1.06E−10 999 Rico/08/34 (H1N1) A/Vietnam/ 0.52 ± 0.01 0.50 3.81E+04 1.99E−05 5.23E−10 580 1203/2004 (H5N1) H1H11729P A/California/ 0.65 ± 0.01 0.84 7.35E+04  1.00E−05* 1.36E−10 1155* 07/2009 (H1N1) A/Puerto 0.67 ± 0.01 0.72 8.26E+04  1.00E−05* 1.21E−10 1155* Rico/08/34 (H1N1) A/Vietnam/ 0.66 ± 0.01 0.62 1.99E+04 3.10E−05 1.56E−09 373 1203/2004 (H5N1) H2aM14571N A/California/ 0.51 ± 0.01 0.58 1.05E+05 1.18E−05 1.12E−10 978 07/2009 (H1N1) A/Puerto  0.5 ± 0.02 0.46 6.80E+04 2.44E−05 3.59E−10 474 Rico/08/34 (H1N1) A/Vietnam/ 0.51 ± 0.01 0.31 1.14E+04  1.00E−05* 8.77E−10 1155* 1203/2004 (H5N1)

Example 4: HA Octet Cross-Competition

Binding competition between a panel of different anti-influenza HA monoclonal antibodies was determined using a real time, label-free bio-layer interferometry assay on an Octet® HTX biosensor (ForteBio, A Division of Pall Life Sciences). The entire experiment was performed at 25° C. in 0.01M HEPES pH7.4, 0.15M NaCl, 0.05% v/v Surfactant Tween-20, and 1 mg/mL BSA (HBS-ET Octet buffer) with the plate shaking at a speed of 1000 rpm. To assess whether two antibodies were able to compete with one another for binding to their respective epitopes on HA, a pre-mix assay format was adopted. One Group-1 strain of HA protein (California) was used to study cross-competition between different anti-HA monoclonal antibodies. To achieve this, 100 nM of HA reagent (Protein Sciences) was first pre-mixed with 1 μM concentration of different anti-HA monoclonal antibodies (subsequently referred to as mAb-2) for at least 2 hours prior to running the binding competition assay. Octet biosensors coated with either an anti-mouse Fc polyclonal antibody (Pall ForteBio Corp., #18-5088; subsequently referred as AMC) or with an anti-human Fc polyclonal antibody (Pall ForteBio Corp., #18-5060; subsequently referred as AHC) were first submerged into wells containing 20 μg/mL solution of individual anti-HA monoclonal antibodies for 3 minutes to capture anti-HA monoclonal antibodies expressed either with a mouse Fc or with a human Fc, respectively (subsequently referred to as mAb-1). Following the capture step, unoccupied anti-mouse Fc polyclonal antibody and anti-human Fc polyclonal antibody on the Octet biosensors were saturated by submerging into wells containing 1 μM solution of a mixture of irrelevant monoclonal antibody expressed with different Fc (human IgG1, mouse IgG2a and mouse IgG2b) for 3 minutes. Finally, the Octet biosensors were immersed for 5 minutes into wells containing the pre-mixed samples of 100 nM of HA and 1 μM concentration of mAb-2. At the end of each cycle, the captured anti-HA monoclonal antibodies along with the bound pre-complex of HA and mAb-2 were regenerated using three alternate 20 second dips into 20 mM H₃PO₄ followed by submerging into HBS-ET Octet buffer. The biosensors were washed in HBS-ET Octet buffer in between every step of the experiment. The real-time binding response was monitored during the entire course of the experiment and the binding response at the end of every step was recorded. During the analysis, the self-self background binding signal (where mAb-1=mAb-2) caused due to the non-specific binding of anti-HA monoclonal antibody to the unoccupied capture surface was subtracted from the entire column and a cross-competition table was generated (Table 4). The response of mAb-1 binding to the pre-complex of HA and mAb-2 was compared and competitive/non-competitive behavior of different anti-HA monoclonal antibodies was determined.

As shown in Table 4, the column on the left shows the mAb1 antibodies that are captured using the AHC Octet biosensors and the column on the right demonstrates the antibodies (mAb2) that cross-compete with the mAb1 antibody.

TABLE 4 Cross-competition between a panel of different anti-HA monoclonal antibodies for binding to H1N1 California strain of HA First mAb (mAb-1) mAb-2 Antibodies Captured using AHC Shown to Compete Octet Biosensors with mAb-1 H2aM11829N H2aM14571N H1H11729P H2aM14571N H2aM11829N H1H11729P H1H11729P H2aM11829N H2aM14571N

Example 4: Selected Group 1-Specific Influenza a Hemagglutinin Monoclonal Antibodies Show Potent Neutralization of Influenza A In Vitro Across Diverse Group 1 Strains

Cell Viability Microneutralization Assay

Monoclonal antibodies were tested in a microneutralization assay to evaluate breadth and potency. In brief, Madin-Darby canine kidney (MDCK) cells were plated at 6.0×10³ cells/well in a 96-well plate. Virus diluent only was added to background control wells. Cells were incubated for 4 to 5 hours at 37° C. with 5% CO₂. Monoclonal antibodies were diluted at 4 times final concentration in virus diluent. The antibodies were diluted 1:3 in duplicate. Virus was thawed on ice and diluted to the appropriate pre-determined concentration. Diluted virus was added to diluted mAbs. The mAb-virus mixture was immediately transferred to the MDCK cells and incubated at 37° C. with 5% CO₂ for 72 h. Virus control and uninfected cell control wells were also included. On day 4, the plates were centrifuged at 1200 RPM for 3 minutes. Cells were lysed using 100 μL CelTiter-Glo substrate and ATP release measured using luminescence (Victor X3, PerkinElmer). Percent viability was determined relative to uninfected control. Viability values were analyzed using non-linear 4PL regression to determine IC₅₀ values (GraphPad Prism).

The mAbs showed potency against a broad range of group 1 influenza A viruses (Table 5), including various H1N1, H5N1, H9N2, H13N6, and H16N3 strains. The IC₅₀ values ranged from 1.68 nM to 48 nM for H2aM11829N and from 2.47 nM to 129.5 nM for H1H11729P.

TABLE 5 Neutralization potency against various representative group 1 Influenza strains. (average of 3 to 5 runs shown) IC₅₀ (nM) PID H1_PR34 H1_WS33 H1_IVR148 H1_CA09 H9_RG26 H13_MD77 H16_DE09 H2aM11829N 3.29 1.68 48.0 3.50 10.2 2.11 7.39 H1H11729P 2.82 2.47 129.5 4.54 14.9 33.8 21.6

Example 5. Selected Group 1-Specific Influenza a Hemagglutinin Monoclonal Antibodies Show Potent Neutralization of Influenza A In Vitro

The exemplary monoclonal antibodies (mAb) H1H11829N2 and H1H11729P were selected for in vitro microneutralization assays. Briefly, Madin-Darby Canine Kidney (MDCK) cells were plated and incubated overnight to achieve 80-100% confluency the next day. Monoclonal antibodies were diluted in viral infection medium (VIM) to 50 pg/mL and diluted 1:2 in triplicate or quadruplicate. H5N1 A/Vietnam/1203/2004 or H1N1 A/California/07/2009 was diluted in VIM and added to the diluted antibodies and incubated for 1 h. The samples were then transferred to the MDCKs and incubated for 48 h. After the incubation, 50 μL of the supernatant was transferred to a new 96-well plate. Diluted turkey or horse red blood cells were added to the supernatant and incubated at room temperature for 30 or 60 min. The hemagglutination titer was recorded as the reciprocal of the last dilution that completely inhibited hemagglutination.

Exemplary antibodies H1H11829N2 and H1H11729P show potent neutralization of H5N1 A/Vietnam/1203/2004 and H1N1 A/California/07/2009 (Table 6). The average IC₉₀ values for exemplary antibodies H1H11829N2 and H1H11729P with respect to neutralization of H5N1 A/Vietnam/1203/2004 were on average 62.50 nM and 26.05 nM (respectively).

TABLE 6 Hemagglutinin titers after microneutralization assay with H5N1 A/Vietnam/1203/2004 and H1N1 A/California/07/2009 with selected group 1 hemagglutinin-specific antibodies. Shown are the IC₉₀ values (nM). IC₉₀ (nM) IC₉₀ (nM) H5N1 H1N1 Antibody A/Vietnam/1203/2004 A/California/07/2009 Number Trial 1 Trial 2 Trial 1 H1H11829N2 83.3 41.7 41.7 H1H11729P 41.7 10.4 83.3

Example 6. Complement-Dependent Cytotoxicity of Influenza Infected Cells In Vitro Using Influenza a Specific Monoclonal Antibodies

The exemplary monoclonal antibodies (mAb) H1H11829N2 and H1H11729P were selected for in vitro complement-dependent cytotoxicity (CDC) assays. Briefly, Madin-Darby Canine Kidney (MDCK) cells were plated 24 hours prior to infection with H1N1 A/Puerto Rico/08/1934 at an MOI of 3. After an incubation of 20 to 25 hours, cells were harvested and resuspended in CDC assay medium at a concentration of 1×10⁶ cells/mL. The diluted target cells (20 μL) were added to each well of a 384-well plate. Monoclonal antibodies were diluted to three times the final starting concentration in CDC assay medium and then diluted 1:2 in triplicate or quadruplicate. CDC assay medium was added to each well not receiving mAb. The plate was shaken at room temperature for ˜2 min at ˜500 rpm. Normal human serum complement (NHSC) was prepared at three times the final concentration (15%) in CDC assay medium and 20 μL was added to each well. The plate was incubated for 2 h at room temperature and the lysis reagent, buffer and substrate was brought to room temperature at this time. The lysis reagent was prepared by dilution of digitonin into CDC assay medium and 5 μL of this reagent was added to the maximal lysis control wells to establish the maximal signal control. The substrate and buffer were combined and 20 μL added to all wells and incubated for 10 min at room temperature. The signal was detected on a plate reader (Victor X3, Perkin Elmer). The percent specific lysis was calculated as ((lysis by mAb+NHSC)−(lysis by NHSC only))/((digitonin maximal lysis)-(lysis by NHSC only))×100. Analysis was completed by a four-parameter non-linear regression over an 8-point response curve (GraphPad Prism). Data was plotted for individual trials±standard deviation, and data presented as averages of all trails±standard error of the mean.

Exemplary antibodies H1H11829N2 and H1H11729P show potent complement-mediated lysis of infected cells. Three randomly selected trials are shown in Table 7. The average EC₅₀ values for exemplary antibodies H1H11829N2 and H1H11729P were on average 41.88 nM (±13.08 SEM) and H1H11729P were 43.25 nM (±3.83 SEM) respectively.

TABLE 7 Percent specific lysis of H1N1 A/Puerto Rico/08/1934-infected target cells by group 1 hemagglutinin-specific antibodies. Shown are the EC₅₀ values (nM) for each of three replicates. EC₅₀ (nM) AbPID Trial 1 Trial 2 Trial 3 H1H11829N2 65.94 38.75 20.94 H1H11729P 50.83 38.49 40.44 hIgG1 irrelevant mAb >333 >333 >333

Example 7: Selected Group 1-Specific Influenza a Hemagglutinin Monoclonal Antibodies Show Potent Neutralization of Influenza a Infection In Vivo

Single Dose Prophylaxis Mouse Model

The exemplary monoclonal antibodies H2aM11829N, H1H11729P and H2a14571N were selected for in vivo protection studies using BALB/c mice. Briefly, approximately 6-week old, female mice (approximately 17.5±0.5 g) were injected subcutaneously (SC) with 1 mg/kg of H2aM11829N, H1H11729P, H2a14571N, or mouse or human isotype (negative control) antibody on day −1. Five mice were included per group per experiment. Twenty-four hours post-injection (day 0), mice were challenged intranasally (IN) with 10×MLD₅₀ (800 plaque forming units; PFUs) in 20 μL saline with H1N1 A/Puerto Rico/08/1934. Mice were weighed daily and were sacrificed when they lost >20% of their initial starting weight.

Exemplary antibodies H2aM11829N, H1H11729P and H2a14571N show potent neutralization of influenza A infection in vivo at 1 mg/kg.

Dose Response Prophylaxis Mouse Model

The exemplary monoclonal antibodies H1H11829N2 and H1H11729P were selected for prophylactic in vivo protection studies using BALB/c mice. Briefly, 6-week old, female mice (approximately 17.5±0.5 g) were injected SC with 1, 0.5, 0.1 or 0.05 mg/kg or 2, 0.5, 0.2 or 0.05 mg/kg of H1H11829N2, H1H11729P antibody on day −1. Five mice were included per group per experiment. Carrier protein (an irrelevant hIgG1 mAb) was added to all doses of mAb at 1 mg/mL. Twenty-four hours post-injection (day 0), mice were challenged intranasally with 10×MLD₅₀ (800 plaque forming units; PFUs) in 20 μL saline with H1N1 A/Puerto Rico/08/1934 or 5×MLD₅₀ (5,000 PFUs) in 20 μL saline with H1N1 A/California/04/2009. Mice were weighed daily and were sacrificed when they lost >20% of their initial starting weight.

When challenged with H1_PR34, exemplary antibodies H1H11829N2 and H1H11729P show potent neutralization of influenza A infection in vivo at 1 mg/kg. At 0.5 and 0.1 mg/kg, H1H11729P shows 100% and 40% survival, respectively. (Tables 8, 9 and 10). When challenged with H1_CA09, exemplary antibodies H1H11829N2 and H1H11729P show potent neutralization of influenza A infection in vivo at 2, 0.5 and 0.2 mg/kg and showed 60% survival at 0.05 mg/kg. (Tables 8, 9 and 10).

TABLE 8 Percent survival of mice in experiment 1 after administration of 1 mg/kg group 1 hemagglutinin-specific antibodies, SC prophylactically one day before infection with 10 × MLD₅₀ of H1N1 A/Puerto Rico/08/1934. Percent survival Number (no. of surviving of mice mice/total no. of PID per group mice in the group) Saline (uninfected) 5 100 (5/5) H2aM11829N 5 100 (5/5) H1H11729P 5 100 (5/5) H2a14571N ND ND hIgG1 isotype control 5 0 (0/5) mIgG2a isotype control 5 0 (0/5)

TABLE 9 Percent survival of mice in experiment 2 after administration of 1 mg/kg group 1 hemagglutinin-specific antibodies, SC prophylactically one day before infection with 10 × MLD₅₀ of H1N1 A/Puerto Rico/08/1934. Percent survival Number (no. of surviving of mice mice/total no. of PID per group mice in the group) Saline (uninfected) 5 100 (5/5) H2aM11829N 5 100 (5/5) H1H11729P 5 100 (5/5) H2a14571N 5 100 (5/5)

TABLE 10 Percent survival of mice after administration of 1, 0.5, 0.1 or 0.05 mg/kg group 1 hemagglutinin-specific antibodies, SC prophylactically one day before infection with 10 x MLD₅₀ of H1N1 A/Puerto Rico/08/1934 or 2, 0.5, 0.2 or 0.05 mg/kg, SC prophylactically one day before infection with 5 x MLD₅₀ of H1N1 A/California/04/2009. Challenge virus: Challenge virus: H1N1 A/Puerto Rico/08/1934 H1N1 A/California/04/2009 Percent survival Percent survival Dose Number (no. of surviving Dose Number (no. of surviving of mAb of mice mice/total no. of of mAb of mice mice/total no. of PID (mg/kg) per group mice in the group) (mg/kg) per group mice in the group) Saline with N/A 5 100 (5/5) N/A 5 100 (5/5) carrier protein (uninfected) H1H11829N2 1 5 100 (5/5) 2 5 100 (5/5) 0.5  60 (3/5) 0.5 100 (5/5) 0.1  0 (0/5) 0.2 100 (5/5) 0.05  20 (1/5) 0.05 ND H1H11729P 1 5 100 (5/5) 2 5 100 (5/5) 0.5 100 (5/5) 0.5 100 (5/5) 0.1  40 (2/5) 0.2 100 (5/5) 0.05  0 (0/5) 0.05  60 (3/5) Dose Response Therapeutic Mouse Model

The exemplary monoclonal antibodies H1H11829N2 and H1H11729P were selected for therapeutic in vivo protection studies using BALB/c mice. Briefly, 6-week old, female mice (approximately 17.5±0.5 g) were infected intranasally with 10×MLD₅₀ (800 plaque forming units; PFUs) in 20 μL saline with H1N1 A/Puerto Rico/08/1934 or 5×MLD₅₀ (5,000 PFUs) in 20 μL saline with H1N1 A/California/04/2009. Three, four or five days (in the case of H1_PR34) or one, two or three days (in the case of H1_CA09) post-infection, separate groups of 5 mice were injected IV with 15 mg/kg or 30 mg/kg of H1H11829N2, or H1H11729P antibody. Mice were weighed daily and were sacrificed when they lost >20% of their initial starting weight.

When challenged with both a historical and contemporary H1N1 virus strain, exemplary antibodies H1H11829N2 and H1H11729P show potent neutralization of influenza A infection in vivo. (Table 11).

TABLE 11 Percent survival of mice in a therapeutic model of infection. Mice were infected with 10 x MLD₅₀ of H1N1 A/Puerto Rico/08/1934 or 5 x MLD₅₀ of H1N1 A/California/04/2009 on day 0. On days 3, 4 or 5 (for H1N1 A/Puerto Rico/08/1934) or days 1, 2 or 3 (for H1N1 A/California/04/2009) mice antibodies were administered at 15 or 30 mg/kg, IV. Challenge virus: Challenge virus: H1N1 A/Puerto Rico/08/1934 H1N1 A/California/04/2009 (15 mg/kg mAb) (30 mg/kg mAb) Percent survival Percent survival Days Number (no. of surviving Days Number (no. of surviving post- of mice mice/total no. of post- of mice mice/total no. of PID infection per group mice in the group) infection per group mice in the group) H1H11829N2 3 5 40 (2/5) 1 5 100 (5/5)  4 ND 2  0 (0/5) 5 ND 3 20 (1/5) H1H11729P 3 5 60 (3/5) 1 5 100 (5/5)   4  0 (0/5) 2  0 (0/5) 5  0 (0/5) 3 20 (1/5)

Example 8. Selected Group 1-Specific Influenza a Hemagglutinin Monoclonal Antibodies are Generated after Immunization with H5 Immunogens

Additional human antibodies to Influenza A-HA were generated in VELOCIMMUNE® mice comprising DNA encoding human immunoglobulin heavy and kappa light chain variable regions. Naïve mice were divided into four groups. Groups 1 and 2 were immunized with trivalent influenza vaccine (TIV) Afluria® 2013-2014 formulation (NDC 3332-110-10). Groups 3 and 4 were left unimmunized. Approximately, four weeks later, groups 1 and 3 were injected intramuscularly with 50 pg H5 DNA (VRC9123 encoding H5 A/Indonesia/05/2005 (see Ledgerwood et al. (2011) Lancet Infect. Dis. 11: 916-924; Khurana et al. (2013) J. Infect. Dis. 208:413-417; Ledgerwood et al. (2013) J. Infect. Dis. 208: 418-422) and groups 2 and 4 were injected intramuscularly with 5 μg H5 monovalent influenza vaccine (MIV; VRC310 containing H5 A/Indonesia/05/2005) (see Ledgerwood et al. (2011) Lancet Infect. Dis. 11: 916-924, Khurana et al. (2013) J. Infect. Dis. 208:413-417; Ledgerwood et al. (2013) J. Infect. Dis. 208: 418-422). Approximately four weeks later, all 4 groups were immunized with 5 μg of H5 monovalent influenza vaccine (MIV; VRC310 containing H5 A/Indonesia/05/2005).

Anti-influenza A-HA antibodies were isolated directly from receptor-binding site (RBS) mutated, avi-tagged H5 A/Indonesia/05/2005 and/or H1 A/New Caledonia/20/1999 that contained a carboxy-terminal trimerization domain from T4 phage fibritin (foldon)-positive mouse B cells without fusion to myeloma cells, as described in U.S. Pat. No. 7,582,298, herein specifically incorporated by reference in its entirety. Using this method, several additional fully human anti-influenza A-HA antibodies (i.e., antibodies possessing human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner can be found in Tables 12 (amino acid sequence identifiers) and 13 (nucleic acid sequence identifiers) below. The samples were evaluated for binding to receptor-binding site (RBS) mutated, foldon, avi-tagged H5 and H1 via Luminex. As expected, all but 16 samples bound to H5 protein with mean fluorescent intensities (MFI) of greater than 15,000. However, for the most part, samples that also bound to H1 protein had high binding to H5 (i.e., greater than 15,000 MFI). Two groups of monoclonal antibodies were observed; those which bound to H1 with greater than 15,000 MFI (20 samples) and those that bound to H5 with greater than 5000 MFI (44 samples).

TABLE 12 Amino Acid Sequence Identifiers Amino Acid SEQ ID NOs: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H17952B 298 300 302 304 306 308 310 312 H1H17953B 314 316 318 320 322 324 326 328 H1H17954B 330 332 334 336 338 340 342 344 H1H17955B 346 348 350 352 354 356 358 360 H1H17956B 362 364 366 368 370 372 374 376 H1H17957B 378 380 382 384 386 388 390 392 H1H17958B 394 396 398 400 402 404 406 408 H1H17959B 410 412 414 416 418 420 422 424 H1H17960B 426 428 430 432 434 436 438 440 H1H17961B 442 444 446 448 450 452 454 456 H1H17962B 458 460 462 464 466 468 470 472 H1H17963B 474 476 478 480 482 484 486 488 H1H17964B 490 492 494 496 498 500 502 504 H1H17965B 506 508 510 512 514 516 518 520 H1H17966B 522 524 526 528 530 532 534 536 H1H17967B 538 540 542 544 546 548 550 552 H1H17968B 554 556 558 560 562 564 566 568 H1H17969B 570 572 574 576 578 580 582 584 H1H17970B 586 588 590 592 594 596 598 600 H1H17971B 602 604 606 608 610 612 614 616 H1H17972B 618 620 622 624 626 628 630 632 H1H17973B 634 636 638 640 642 644 646 648 H1H17974B 650 652 654 656 658 660 662 664 H1H17975B 666 668 670 672 674 676 678 680 H1H17976B 682 684 686 688 690 692 694 696 H1H17977B 698 700 702 704 706 708 710 712 H1H17978B 714 716 718 720 722 724 726 728 H1H17979B 730 732 734 736 738 740 742 744 H1H17980B 746 748 750 752 754 756 758 760 H1H17981B 762 764 766 768 770 772 774 776 H1H17982B 778 780 782 784 786 788 790 792 H1H17983B 794 796 798 800 802 804 806 808 H1H17984B 810 812 814 816 818 820 822 824 H1H17985B 826 828 830 832 834 836 838 840 H1H17986B 842 844 846 848 850 852 854 856 H1H17987B 858 860 862 864 866 868 870 872 H1H17988B 874 876 878 880 882 884 886 888 H1H17989B 890 892 894 896 898 900 902 904 H1H17990B 906 908 910 912 914 916 918 920 H1H17991B 922 924 926 928 930 932 934 936 H1H17992B 938 940 942 944 946 948 950 952 H1H17993B 954 956 958 960 962 964 966 968 H1H17994B 970 972 974 976 978 980 982 984 H1H17995B 986 988 990 992 994 996 998 1000 H1H17996B 1002 1004 1006 1008 1010 1012 1014 1016 H1H17997B 1018 1020 1022 1024 1026 1028 1030 1032 H1H17998B 1034 1036 1038 1040 1042 1044 1046 1048 H1H17999B 1050 1052 1054 1056 1058 1060 1062 1064 H1H18000B 1066 1068 1070 1072 1074 1076 1078 1080 H1H18001B 1082 1084 1086 1088 1090 1092 1094 1096 H1H18002B 1098 1100 1102 1104 1106 1108 1110 1112 H1H18003B 1114 1116 1118 1120 1122 1124 1126 1128 H1H18004B 1130 1132 1134 1136 1138 1140 1142 1144 H1H18005B 1146 1148 1150 1152 1154 1156 1158 1160 H1H18006B 1162 1164 1166 1168 1170 1172 1174 1176 H1H18007B 1178 1180 1182 1184 1186 1188 1190 1192 H1H18008B 1194 1196 1198 1200 1202 1204 1206 1208 H1H18009B 1210 1212 1214 1216 1218 1220 1222 1224 H1H18010B 1226 1228 1230 1232 1234 1236 1238 1240 H1H18011B 1242 1244 1246 1248 1250 1252 1254 1256 H1H18012B 1258 1260 1262 1264 1266 1268 1270 1272 H1H18013B 1274 1276 1278 1280 1282 1284 1286 1288 H1H18014B 1290 1292 1294 1296 1298 1300 1302 1304 H1H18015B 1306 1308 1310 1312 1314 1316 1318 1320 H1H18016B 1322 1324 1326 1328 1330 1332 1334 1336 H1H18017B 1338 1340 1342 1344 1346 1348 1350 1352 H1H18018B 1354 1356 1358 1360 1362 1364 1366 1368 H1H18019B 1370 1372 1374 1376 1378 1380 1382 1384 H1H18020B 1386 1388 1390 1392 1394 1396 1398 1400 H1H18021B 1402 1404 1406 1408 1410 1412 1414 1416 H1H18022B 1418 1420 1422 1424 1426 1428 1430 1432 H1H18023B 1434 1436 1438 1440 1442 1444 1446 1448 H1H18024B 1450 1452 1454 1456 1458 1460 1462 1464 H1H18025B 1466 1468 1470 1472 1474 1476 1478 1480 H1H18026B 1482 1484 1486 1488 1490 1492 1494 1496 H1H18027B 1498 1500 1502 1504 1506 1508 1510 1512 H1H18028B 1514 1516 1518 1520 1522 1524 1526 1528 H1H18029B 1530 1532 1534 1536 1538 1540 1542 1544 H1H18030B 1546 1548 1550 1552 1554 1556 1558 1560 H1H18031B 1562 1564 1566 1568 1570 1572 1574 1576 H1H18032B 1578 1580 1582 1584 1586 1588 1590 1592 H1H18033B 1594 1596 1598 1600 1602 1604 1606 1608 H1H18034B 1610 1612 1614 1616 1618 1620 1622 1624 H1H18035B 1626 1628 1630 1632 1634 1636 1638 1640 H1H18037B 1642 1644 1646 1648 1650 1652 1654 1656 H1H18038B 1658 1660 1662 1664 1666 1668 1670 1672 H1H18039B 1674 1676 1678 1680 1682 1684 1686 1688 H1H18040B 1690 1692 1694 1696 1698 1700 1702 1704 H1H18041B 1706 1708 1710 1712 1714 1716 1718 1720 H1H18042B 1722 1724 1726 1728 1730 1732 1734 1736 H1H18043B 1738 1740 1742 1744 1746 1748 1750 1752 H1H18044B 1754 1756 1758 1760 1762 1764 1766 1768 H1H18045B 1770 1772 1774 1776 1778 1780 1782 1784 H1H18046B 1786 1788 1790 1792 1794 1796 1798 1800 H1H18047B 1802 1804 1806 1808 1810 1812 1814 1816 H1H18048B 1818 1820 1822 1824 1826 1828 1830 1832 H1H18049B 1834 1836 1838 1840 1842 1844 1846 1848 H1H18051B 1850 1852 1854 1856 1858 1860 1862 1864 H1H18052B 1866 1868 1870 1872 1874 1876 1878 1880 H1H18053B 1882 1884 1886 1888 1890 1892 1894 1896 H1H18054B 1898 1900 1902 1904 1906 1908 1910 1912 H1H18055B 1914 1916 1918 1920 1922 1924 1926 1928 H1H18056B 1930 1932 1934 1936 1938 1940 1942 1944 H1H18057B 1946 1948 1950 1952 1954 1956 1958 1960 H1H18058B 1962 1964 1966 1968 1970 1972 1974 1976 H1H18059B 1978 1980 1982 1984 1986 1988 1990 1992 H1H18060B 1994 1996 1998 2000 2002 2004 2006 2008 H1H18061B 2010 2012 2014 2016 2018 2020 2022 2024 H1H18062B 2026 2028 2030 2032 2034 2036 2038 2040 H1H18063B 2042 2044 2046 2048 2050 2052 2054 2056 H1H18064B 2058 2060 2062 2064 2066 2068 2070 2072 H1H18065B 2074 2076 2078 2080 2082 2084 2086 2088 H1H18066B 2090 2092 2094 2096 2098 2100 2102 2104 H1H18067B 2106 2108 2110 2112 2114 2116 2118 2120 H1H18068B 2122 2124 2126 2128 2130 2132 2134 2136 H1H18069B 2138 2140 2142 2144 2146 2148 2150 2152 H1H18070B 2154 2156 2158 2160 2162 2164 2166 2168 H1H18071B 2170 2172 2174 2176 2178 2180 2182 2184 H1H18072B 2186 2188 2190 2192 2194 2196 2198 2200 H1H18073B 2202 2204 2206 2208 2210 2212 2214 2216 H1H18074B 2218 2220 2222 2224 2226 2228 2230 2232 H1H18075B 2234 2236 2238 2240 2242 2244 2246 2248 H1H18076B 2250 2252 2254 2256 2258 2260 2262 2264 H1H18077B 2266 2268 2270 2272 2274 2276 2278 2280 H1H18078B 2282 2284 2286 2288 2290 2292 2294 2296 H1H18079B 2298 2300 2302 2304 2306 2308 2310 2312 H1H18080B 2314 2316 2318 2320 2322 2324 2326 2328 H1H18081B 2330 2332 2334 2336 2338 2340 2342 2344 H1H18082B 2346 2348 2350 2352 2354 2356 2358 2360 H1H18083B 2362 2364 2366 2368 2370 2372 2374 2376 H1H18084B 2378 2380 2382 2384 2386 2388 2390 2392 H1H18085B 2394 2396 2398 2400 2402 2404 2406 2408 H1H18086B 2410 2412 2414 2416 2418 2420 2422 2424 H1H18087B 2426 2428 2430 2432 2434 2436 2438 2440 H1H18088B 2442 2444 2446 2448 2450 2452 2454 2456 H1H18089B 2458 2460 2462 2464 2466 2468 2470 2472 H1H18090B 2474 2476 2478 2480 2482 2484 2486 2488 H1H18091B 2490 2492 2494 2496 2498 2500 2502 2504 H1H18092B 2506 2508 2510 2512 2514 2516 2518 2520 H1H18093B 2522 2524 2526 2528 2530 2532 2534 2536 H1H18094B 2538 2540 2542 2544 2546 2548 2550 2552 H1H18095B 2554 2556 2558 2560 2562 2564 2566 2568 H1H18096B 2570 2572 2574 2576 2578 2580 2582 2584 H1H18097B 2586 2588 2590 2592 2594 2596 2598 2600 H1H18098B 2602 2604 2606 2608 2610 2612 2614 2616 H1H18099B 2618 2620 2622 2624 2626 2628 2630 2632 H1H18100B 2634 2636 2638 2640 2642 2644 2646 2648 H1H18101B 2650 2652 2654 2656 2658 2660 2662 2664 H1H18102B 2666 2668 2670 2672 2674 2676 2678 2680 H1H18103B 2682 2684 2686 2688 2690 2692 2694 2696 H1H18104B 2698 2700 2702 2704 2706 2708 2710 2712 H1H18105B 2714 2716 2718 2720 2722 2724 2726 2728 H1H18107B 2730 2732 2734 2736 2738 2740 2742 2744 H1H18108B 2746 2748 2750 2752 2754 2756 2758 2760 H1H18109B 2762 2764 2766 2768 2770 2772 2774 2776 H1H18110B 2778 2780 2782 2784 2786 2788 2790 2792 H1H18111B 2794 2796 2798 2800 2802 2804 2806 2808 H1H18112B 2810 2812 2814 2816 2818 2820 2822 2824 H1H18113B 2826 2828 2830 2832 2834 2836 2838 2840 H1H18114B 2842 2844 2846 2848 2850 2852 2854 2856 H1H18115B 2858 2860 2862 2864 2866 2868 2870 2872 H1H18116B 2874 2876 2878 2880 2882 2884 2886 2888 H1H18117B 2890 2892 2894 2896 2898 2900 2902 2904 H1H18118B 2906 2908 2910 2912 2914 2916 2918 2920 H1H18119B 2922 2924 2926 2928 2930 2932 2934 2936 H1H18120B 2938 2940 2942 2944 2946 2948 2950 2952 H1H18121B 2954 2956 2958 2960 2962 2964 2966 2968 H1H18122B 2970 2972 2974 2976 2978 2980 2982 2984 H1H18123B 2986 2988 2990 2992 2994 2996 2998 3000 H1H18124B 3002 3004 3006 3008 3010 3012 3014 3016 H1H18125B 3018 3020 3022 3024 3026 3028 3030 3032 H1H18126B 3034 3036 3038 3040 3042 3044 3046 3048 H1H18127B 3050 3052 3054 3056 3058 3060 3062 3064 H1H18128B 3066 3068 3070 3072 3074 3076 3078 3080 H1H18129B 3082 3084 3086 3088 3090 3092 3094 3096 H1H18130B 3098 3100 3102 3104 3106 3108 3110 3112 H1H18131B 3114 3116 3118 3120 3122 3124 3126 3128 H1H18132B 3130 3132 3134 3136 3138 3140 3142 3144 H1H18133B 3146 3148 3150 3152 3154 3156 3158 3160 H1H18134B 3162 3164 3166 3168 3170 3172 3174 3176 H1H18135B 3178 3180 3182 3184 3186 3188 3190 3192 H1H18136B 3194 3196 3198 3200 3202 3204 3206 3208 H1H18137B 3210 3212 3214 3216 3218 3220 3222 3224 H1H18138B 3226 3228 3230 3232 3234 3236 3238 3240 H1H18139B 3242 3244 3246 3248 3250 3252 3254 3256 H1H18140B 3258 3260 3262 3264 3266 3268 3270 3272 H1H18141B 3274 3276 3278 3280 3282 3284 3286 3288 H1H18142B 3290 3292 3294 3296 3298 3300 3302 3304 H1H18143B 3306 3308 3310 3312 3314 3316 3318 3320 H1H18144B 3322 3324 3326 3328 3330 3332 3334 3336 H1H18145B 3338 3340 3342 3344 3346 3348 3350 3352 H1H18146B 3354 3356 3358 3360 3362 3364 3366 3368 H1H18147B 3370 3372 3374 3376 3378 3380 3382 3384 H1H18148B 3386 3388 3390 3392 3394 3396 3398 3400 H1H18149B 3402 3404 3406 3408 3410 3412 3414 3416 H1H18150B 3418 3420 3422 3424 3426 3428 3430 3432 H1H18151B 3434 3436 3438 3440 3442 3444 3446 3448 H1H18152B 3450 3452 3454 3456 3458 3460 3462 3464 H1H18153B 3466 3468 3470 3472 3474 3476 3478 3480 H1H18154B 3482 3484 3486 3488 3490 3492 3494 3496 H1H18155B 3498 3500 3502 3504 3506 3508 3510 3512 H1H18156B 3514 3516 3518 3520 3522 3524 3526 3528 H1H18157B 3530 3532 3534 3536 3538 3540 3542 3544 H1H18158B 3546 3548 3550 3552 3554 3556 3558 3560 H1H18159B 3562 3564 3566 3568 3570 3572 3574 3576 H1H18160B 3578 3580 3582 3584 3586 3588 3590 3592 H1H18161B 3594 3596 3598 3600 3602 3604 3606 3608 H1H18162B 3610 3612 3614 3616 3618 3620 3622 3624 H1H18163B 3626 3628 3630 3632 3634 3636 3638 3640 H1H18164B 3642 3644 3646 3648 3650 3652 3654 3656 H1H18165B 3658 3660 3662 3664 3666 3668 3670 3672 H1H18166B 3674 3676 3678 3680 3682 3684 3686 3688 H1H18167B 3690 3692 3694 3696 3698 3700 3702 3704 H1H18168B 3706 3708 3710 3712 3714 3716 3718 3720 H1H18169B 3722 3724 3726 3728 3730 3732 3734 3736 H1H18170B 3738 3740 3742 3744 3746 3748 3750 3752 H1H18171B 3754 3756 3758 3760 3762 3764 3766 3768 H1H18172B 3770 3772 3774 3776 3778 3780 3782 3784 H1H18173B 3786 3788 3790 3792 3794 3796 3798 3800 H1H18174B 3802 3804 3806 3808 3810 3812 3814 3816 H1H18175B 3818 3820 3822 3824 3826 3828 3830 3832 H1H18176B 3834 3836 3838 3840 3842 3844 3846 3848 H1H18177B 3850 3852 3854 3856 3858 3860 3862 3864 H1H18178B 3866 3868 3870 3872 3874 3876 3878 3880 H1H18179B 3882 3884 3886 3888 3890 3892 3894 3896 H1H18180B 3898 3900 3902 3904 3906 3908 3910 3912 H1H18181B 3914 3916 3918 3920 3922 3924 3926 3928 H1H18182B 3930 3932 3934 3936 3938 3940 3942 3944 H1H18183B 3946 3948 3950 3952 3954 3956 3958 3960 H1H18184B 3962 3964 3966 3968 3970 3972 3974 3976 H1H18185B 3978 3980 3982 3984 3986 3988 3990 3992 H1H18186B 3994 3996 3998 4000 4002 4004 4006 4008 H1H18187B 4010 4012 4014 4016 4018 4020 4022 4024 H1H18188B 4026 4028 4030 4032 4034 4036 4038 4040 H1H18189B 4042 4044 4046 4048 4050 4052 4054 4056 H1H18190B 4058 4060 4062 4064 4066 4068 4070 4072 H1H18191B 4074 4076 4078 4080 4082 4084 4086 4088 H1H18192B 4090 4092 4094 4096 4098 4100 4102 4104 H1H18193B 4106 4108 4110 4112 4114 4116 4118 4120 H1H18194B 4122 4124 4126 4128 4130 4132 4134 4136 H1H18195B 4138 4140 4142 4144 4146 4148 4150 4152 H1H18196B 4154 4156 4158 4160 4162 4164 4166 4168 H1H18197B 4170 4172 4174 4176 4178 4180 4182 4184 H1H18198B 4186 4188 4190 4192 4194 4196 4198 4200 H1H18199B 4202 4204 4206 4208 4210 4212 4214 4216 H1H18200B 4218 4220 4222 4224 4226 4228 4230 4232 H1H18201B 4234 4236 4238 4240 4242 4244 4246 4248 H1H18202B 4250 4252 4254 4256 4258 4260 4262 4264 H1H18203B 4266 4268 4270 4272 4274 4276 4278 4280 H1H18204B 4282 4284 4286 4288 4290 4292 4294 4296 H1H18205B 4298 4300 4302 4304 4306 4308 4310 4312 H1H18206B 4314 4316 4318 4320 4322 4324 4326 4328 H1H18207B 4330 4332 4334 4336 4338 4340 4342 4344 H1H18208B 4346 4348 4350 4352 4354 4356 4358 4360 H1H18209B 4362 4364 4366 4368 4370 4372 4374 4376 H1H18210B 4378 4380 4382 4384 4386 4388 4390 4392 H1H18211B 4394 4396 4398 4400 4402 4404 4406 4408 H1H18212B 4410 4412 4414 4416 4418 4420 4422 4424 H1H18213B 4426 4428 4430 4432 4434 4436 4438 4440 H1H18214B 4442 4444 4446 4448 4450 4452 4454 4456 H1H18216B 4458 4460 4462 4464 4466 4468 4470 4472 H1H18217B 4474 4476 4478 4480 4482 4484 4486 4488 H1H18218B 4490 4492 4494 4496 4498 4500 4502 4504 H1H18219B 4506 4508 4510 4512 4514 4516 4518 4520 H1H18220B 4522 4524 4526 4528 4530 4532 4534 4536 H1H18221B 4538 4540 4542 4544 4546 4548 4550 4552 H1H18222B 4554 4556 4558 4560 4562 4564 4566 4568 H1H18223B 4570 4572 4574 4576 4578 4580 4582 4584 H1H18224B 4586 4588 4590 4592 4594 4596 4598 4600 H1H18225B 4602 4604 4606 4608 4610 4612 4614 4616 H1H18226B 4618 4620 4622 4624 4626 4628 4630 4632 H1H18227B 4634 4636 4638 4640 4642 4644 4646 4648 H1H18228B 4650 4652 4654 4656 4658 4660 4662 4664 H1H18229B 4666 4668 4670 4672 4674 4676 4678 4680 H1H18230B 4682 4684 4686 4688 4690 4692 4694 4696 H1H18231B 4698 4700 4702 4704 4706 4708 4710 4712 H1H18232B 4714 4716 4718 4720 4722 4724 4726 4728 H1H18233B 4730 4732 4734 4736 4738 4740 4742 4744 H1H18234B 4746 4748 4750 4752 4754 4756 4758 4760 H1H18235B 4762 4764 4766 4768 4770 4772 4774 4776 H1H18236B 4778 4780 4782 4784 4786 4788 4790 4792 H1H18237B 4794 4796 4798 4800 4802 4804 4806 4808 H1H18238B 4810 4812 4814 4816 4818 4820 4822 4824 H1H18239B 4826 4828 4830 4832 4834 4836 4838 4840 H1H18240B 4842 4844 4846 4848 4850 4852 4854 4856 H1H18241B 4858 4860 4862 4864 4866 4868 4870 4872 H1H18242B 4874 4876 4878 4880 4882 4884 4886 4888 H1H18243B 4890 4892 4894 4896 4898 4900 4902 4904 H1H18244B 4906 4908 4910 4912 4914 4916 4918 4920 H1H18245B 4922 4924 4926 4928 4930 4932 4934 4936 H1H18246B 4938 4940 4942 4944 4946 4948 4950 4952 H1H18247B 4954 4956 4958 4960 4962 4964 4966 4968 H1H18248B 4970 4972 4974 4976 4978 4980 4982 4984 H1H18249B 4986 4988 4990 4992 4994 4996 4998 5000 H1H18250B 5002 5004 5006 5008 5010 5012 5014 5016 H1H18251B 5018 5020 5022 5024 5026 5028 5030 5032 H1H18252B 5034 5036 5038 5040 5042 5044 5046 5048 H1H18253B 5050 5052 5054 5056 5058 5060 5062 5064 H1H18254B 5066 5068 5070 5072 5074 5076 5078 5080 H1H18255B 5082 5084 5086 5088 5090 5092 5094 5096 H1H18256B 5098 5100 5102 5104 5106 5108 5110 5112 H1H18257B 5114 5116 5118 5120 5122 5124 5126 5128 H1H18258B 5130 5132 5134 5136 5138 5140 5142 5144 H1H18259B 5146 5148 5150 5152 5154 5156 5158 5160 H1H18261B 5162 5164 5166 5168 5170 5172 5174 5176 H1H18262B 5178 5180 5182 5184 5186 5188 5190 5192 H1H18263B 5194 5196 5198 5200 5202 5204 5206 5208 H1H18264B 5210 5212 5214 5216 5218 5220 5222 5224 H1H18265B 5226 5228 5230 5232 5234 5236 5238 5240 H1H18266B 5242 5244 5246 5248 5250 5252 5254 5256 H1H18267B 5258 5260 5262 5264 5266 5268 5270 5272 H1H18268B 5274 5276 5278 5280 5282 5284 5286 5288 H1H18269B 5290 5292 5294 5296 5298 5300 5302 5304 H1H18270B 5306 5308 5310 5312 5314 5316 5318 5320 H1H18271B 5322 5324 5326 5328 5330 5332 5334 5336 H1H18272B 5338 5340 5342 5344 5346 5348 5350 5352 H1H18274B 5354 5356 5358 5360 5362 5364 5366 5368 H1H18275B 5370 5372 5374 5376 5378 5380 5382 5384 H1H18276B 5386 5388 5390 5392 5394 5396 5398 5400 H1H18277B 5402 5404 5406 5408 5410 5412 5414 5416 H1H18278B 5418 5420 5422 5424 5426 5428 5430 5432 H1H18279B 5434 5436 5438 5440 5442 5444 5446 5448 H1H18280B 5450 5452 5454 5456 5458 5460 5462 5464 H1H18281B 5466 5468 5470 5472 5474 5476 5478 5480 H1H18282B 5482 5484 5486 5488 5490 5492 5494 5496 H1H18283B 5498 5500 5502 5504 5506 5508 5510 5512 H1H18284B 5514 5516 5518 5520 5522 5524 5526 5528 H1H18285B 5530 5532 5534 5536 5538 5540 5542 5544 H1H18286B 5546 5548 5550 5552 5554 5556 5558 5560 H1H18287B 5562 5564 5566 5568 5570 5572 5574 5576 H1H18288B 5578 5580 5582 5584 5586 5588 5590 5592 H1H18289B 5594 5596 5598 5600 5602 5604 5606 5608 H1H18290B 5610 5612 5614 5616 5618 5620 5622 5624 H1H18291B 5626 5628 5630 5632 5634 5636 5638 5640 H1H18292B 5642 5644 5646 5648 5650 5652 5654 5656 H1H18293B 5658 5660 5662 5664 5666 5668 5670 5672 H1H18294B 5674 5676 5678 5680 5682 5684 5686 5688 H1H18295B 5690 5692 5694 5696 5698 5700 5702 5704 H1H18297B 5706 5708 5710 5712 5714 5716 5718 5720 H1H18298B 5722 5724 5726 5728 5730 5732 5734 5736 H1H18299B 5738 5740 5742 5744 5746 5748 5750 5752 H1H18300B 5754 5756 5758 5760 5762 5764 5766 5768 H1H18301B 5770 5772 5774 5776 5778 5780 5782 5784 H1H18302B 5786 5788 5790 5792 5794 5796 5798 5800 H1H18303B 5802 5804 5806 5808 5810 5812 5814 5816 H1H18304B 5818 5820 5822 5824 5826 5828 5830 5832 H1H18305B 5834 5836 5838 5840 5842 5844 5846 5848 H1H18306B 5850 5852 5854 5856 5858 5860 5862 5864 H1H18307B 5866 5868 5870 5872 5874 5876 5878 5880 H1H18308B 5882 5884 5886 5888 5890 5892 5894 5896 H1H18309B 5898 5900 5902 5904 5906 5908 5910 5912 H1H18310B 5914 5916 5918 5920 5922 5924 5926 5928 H1H18311B 5930 5932 5934 5936 5938 5940 5942 5944 H1H18312B 5946 5948 5950 5952 5954 5956 5958 5960 H1H18313B 5962 5964 5966 5968 5970 5972 5974 5976 H1H18314B 5978 5980 5982 5984 5986 5988 5990 5992 H1H18315B 5994 5996 5998 6000 6002 6004 6006 6008 H1H18316B 6010 6012 6014 6016 6018 6020 6022 6024 H1H18317B 6026 6028 6030 6032 6034 6036 6038 6040 H1H18318B 6042 6044 6046 6048 6050 6052 6054 6056 H1H18319B 6058 6060 6062 6064 6066 6068 6070 6072 H1H18320B 6074 6076 6078 6080 6082 6084 6086 6088 H1H18321B 6090 6092 6094 6096 6098 6100 6102 6104 H1H18322B 6106 6108 6110 6112 6114 6116 6118 6120 H1H18323B 6122 6124 6126 6128 6130 6132 6134 6136 H1H18324B 6138 6140 6142 6144 6146 6148 6150 6152 H1H18325B 6154 6156 6158 6160 6162 6164 6166 6168 H1H18326B 6170 6172 6174 6176 6178 6180 6182 6184 H1H18327B 6186 6188 6190 6192 6194 6196 6198 6200 H1H18328B 6202 6204 6206 6208 6210 6212 6214 6216 H1H18329B 6218 6220 6222 6224 6226 6228 6230 6232 H1H18330B 6234 6236 6238 6240 6242 6244 6246 6248 H1H18331B 6250 6252 6254 6256 6258 6260 6262 6264 H1H18332B 6266 6268 6270 6272 6274 6276 6278 6280 H1H18333B 6282 6284 6286 6288 6290 6292 6294 6296 H1H18334B 6298 6300 6302 6304 6306 6308 6310 6312 H1H18335B 6314 6316 6318 6320 6322 6324 6326 6328

TABLE 13 Nucleic Acid Sequence Identifiers Nucleic Acid SEQ ID NOs: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H17952B 297 299 301 303 305 307 309 311 H1H17953B 313 315 317 319 321 323 325 327 H1H17954B 329 331 333 335 337 339 341 343 H1H17955B 345 347 349 351 353 355 357 359 H1H17956B 361 363 365 367 369 371 373 375 H1H17957B 377 379 381 383 385 387 389 391 H1H17958B 393 395 397 399 401 403 405 407 H1H17959B 409 411 413 415 417 419 421 423 H1H17960B 425 427 429 431 433 435 437 439 H1H17961B 441 443 445 447 449 451 453 455 H1H17962B 457 459 461 463 465 467 469 471 H1H17963B 473 475 477 479 481 483 485 487 H1H17964B 489 491 493 495 497 499 501 503 H1H17965B 505 507 509 511 513 515 517 519 H1H17966B 521 523 525 527 529 531 533 535 H1H17967B 537 539 541 543 545 547 549 551 H1H17968B 553 555 557 559 561 563 565 567 H1H17969B 569 571 573 575 577 579 581 583 H1H17970B 585 587 589 591 593 595 597 599 H1H17971B 601 603 605 607 609 611 613 615 H1H17972B 617 619 621 623 625 627 629 631 H1H17973B 633 635 637 639 641 643 645 647 H1H17974B 649 651 653 655 657 659 661 663 H1H17975B 665 667 669 671 673 675 677 679 H1H17976B 681 683 685 687 689 691 693 695 H1H17977B 697 699 701 703 705 707 709 711 H1H17978B 713 715 717 719 721 723 725 727 H1H17979B 729 731 733 735 737 739 741 743 H1H17980B 745 747 749 751 753 755 757 759 H1H17981B 761 763 765 767 769 771 773 775 H1H17982B 111 779 781 783 785 787 789 791 H1H17983B 793 795 797 799 801 803 805 807 H1H17984B 809 811 813 815 817 819 821 823 H1H17985B 825 827 829 831 833 835 837 839 H1H17986B 841 843 845 847 849 851 853 855 H1H17987B 857 859 861 863 865 867 869 871 H1H17988B 873 875 877 879 881 883 885 887 H1H17989B 889 891 893 895 897 899 901 903 H1H17990B 905 907 909 911 913 915 917 919 H1H17991B 921 923 925 927 929 931 933 935 H1H17992B 937 939 941 943 945 947 949 951 H1H17993B 953 955 957 959 961 963 965 967 H1H17994B 969 971 973 975 977 979 981 983 H1H17995B 985 987 989 991 993 995 997 999 H1H17996B 1001 1003 1005 1007 1009 1011 1013 1015 H1H17997B 1017 1019 1021 1023 1025 1027 1029 1031 H1H17998B 1033 1035 1037 1039 1041 1043 1045 1047 H1H17999B 1049 1051 1053 1055 1057 1059 1061 1063 H1H18000B 1065 1067 1069 1071 1073 1075 1077 1079 H1H18001B 1081 1083 1085 1087 1089 1091 1093 1095 H1H18002B 1097 1099 1101 1103 1105 1107 1109 1111 H1H18003B 1113 1115 1117 1119 1121 1123 1125 1127 H1H18004B 1129 1131 1133 1135 1137 1139 1141 1143 H1H18005B 1145 1147 1149 1151 1153 1155 1157 1159 H1H18006B 1161 1163 1165 1167 1169 1171 1173 1175 H1H18007B 1177 1179 1181 1183 1185 1187 1189 1191 H1H18008B 1193 1195 1197 1199 1201 1203 1205 1207 H1H18009B 1209 1211 1213 1215 1217 1219 1221 1223 H1H18010B 1225 1227 1229 1231 1233 1235 1237 1239 H1H18011B 1241 1243 1245 1247 1249 1251 1253 1255 H1H18012B 1257 1259 1261 1263 1265 1267 1269 1271 H1H18013B 1273 1275 1277 1279 1281 1283 1285 1287 H1H18014B 1289 1291 1293 1295 1297 1299 1301 1303 H1H18015B 1305 1307 1309 1311 1313 1315 1317 1319 H1H18016B 1321 1323 1325 1327 1329 1331 1333 1335 H1H18017B 1337 1339 1341 1343 1345 1347 1349 1351 H1H18018B 1353 1355 1357 1359 1361 1363 1365 1367 H1H18019B 1369 1371 1373 1375 1377 1379 1381 1383 H1H18020B 1385 1387 1389 1391 1393 1395 1397 1399 H1H18021B 1401 1403 1405 1407 1409 1411 1413 1415 H1H18022B 1417 1419 1421 1423 1425 1427 1429 1431 H1H18023B 1433 1435 1437 1439 1441 1443 1445 1447 H1H18024B 1449 1451 1453 1455 1457 1459 1461 1463 H1H18025B 1465 1467 1469 1471 1473 1475 1477 1479 H1H18026B 1481 1483 1485 1487 1489 1491 1493 1495 H1H18027B 1497 1499 1501 1503 1505 1507 1509 1511 H1H18028B 1513 1515 1517 1519 1521 1523 1525 1527 H1H18029B 1529 1531 1533 1535 1537 1539 1541 1543 H1H18030B 1545 1547 1549 1551 1553 1555 1557 1559 H1H18031B 1561 1563 1565 1567 1569 1571 1573 1575 H1H18032B 1577 1579 1581 1583 1585 1587 1589 1591 H1H18033B 1593 1595 1597 1599 1601 1603 1605 1607 H1H18034B 1609 1611 1613 1615 1617 1619 1621 1623 H1H18035B 1625 1627 1629 1631 1633 1635 1637 1639 H1H18037B 1641 1643 1645 1647 1649 1651 1653 1655 H1H18038B 1657 1659 1661 1663 1665 1667 1669 1671 H1H18039B 1673 1675 1677 1679 1681 1683 1685 1687 H1H18040B 1689 1691 1693 1695 1697 1699 1701 1703 H1H18041B 1705 1707 1709 1711 1713 1715 1717 1719 H1H18042B 1721 1723 1725 1727 1729 1731 1733 1735 H1H18043B 1737 1739 1741 1743 1745 1747 1749 1751 H1H18044B 1753 1755 1757 1759 1761 1763 1765 1767 H1H18045B 1769 1771 1773 1775 1777 1779 1781 1783 H1H18046B 1785 1787 1789 1791 1793 1795 1797 1799 H1H18047B 1801 1803 1805 1807 1809 1811 1813 1815 H1H18048B 1817 1819 1821 1823 1825 1827 1829 1831 H1H18049B 1833 1835 1837 1839 1841 1843 1845 1847 H1H18051B 1849 1851 1853 1855 1857 1859 1861 1863 H1H18052B 1865 1867 1869 1871 1873 1875 1877 1879 H1H18053B 1881 1883 1885 1887 1889 1891 1893 1895 H1H18054B 1897 1899 1901 1903 1905 1907 1909 1911 H1H18055B 1913 1915 1917 1919 1921 1923 1925 1927 H1H18056B 1929 1931 1933 1935 1937 1939 1941 1943 H1H18057B 1945 1947 1949 1951 1953 1955 1957 1959 H1H18058B 1961 1963 1965 1967 1969 1971 1973 1975 H1H18059B 1977 1979 1981 1983 1985 1987 1989 1991 H1H18060B 1993 1995 1997 1999 2001 2003 2005 2007 H1H18061B 2009 2011 2013 2015 2017 2019 2021 2023 H1H18062B 2025 2027 2029 2031 2033 2035 2037 2039 H1H18063B 2041 2043 2045 2047 2049 2051 2053 2055 H1H18064B 2057 2059 2061 2063 2065 2067 2069 2071 H1H18065B 2073 2075 2077 2079 2081 2083 2085 2087 H1H18066B 2089 2091 2093 2095 2097 2099 2101 2103 H1H18067B 2105 2107 2109 2111 2113 2115 2117 2119 H1H18068B 2121 2123 2125 2127 2129 2131 2133 2135 H1H18069B 2137 2139 2141 2143 2145 2147 2149 2151 H1H18070B 2153 2155 2157 2159 2161 2163 2165 2167 H1H18071B 2169 2171 2173 2175 2177 2179 2181 2183 H1H18072B 2185 2187 2189 2191 2193 2195 2197 2199 H1H18073B 2201 2203 2205 2207 2209 2211 2213 2215 H1H18074B 2217 2219 2221 2223 2225 2227 2229 2231 H1H18075B 2233 2235 2237 2239 2241 2243 2245 2247 H1H18076B 2249 2251 2253 2255 2257 2259 2261 2263 H1H18077B 2265 2267 2269 2271 2273 2275 2277 2279 H1H18078B 2281 2283 2285 2287 2289 2291 2293 2295 H1H18079B 2297 2299 2301 2303 2305 2307 2309 2311 H1H18080B 2313 2315 2317 2319 2321 2323 2325 2327 H1H18081B 2329 2331 2333 2335 2337 2339 2341 2343 H1H18082B 2345 2347 2349 2351 2353 2355 2357 2359 H1H18083B 2361 2363 2365 2367 2369 2371 2373 2375 H1H18084B 2377 2379 2381 2383 2385 2387 2389 2391 H1H18085B 2393 2395 2397 2399 2401 2403 2405 2407 H1H18086B 2409 2411 2413 2415 2417 2419 2421 2423 H1H18087B 2425 2427 2429 2431 2433 2435 2437 2439 H1H18088B 2441 2443 2445 2447 2449 2451 2453 2455 H1H18089B 2457 2459 2461 2463 2465 2467 2469 2471 H1H18090B 2473 2475 2477 2479 2481 2483 2485 2487 H1H18091B 2489 2491 2493 2495 2497 2499 2501 2503 H1H18092B 2505 2507 2509 2511 2513 2515 2517 2519 H1H18093B 2521 2523 2525 2527 2529 2531 2533 2535 H1H18094B 2537 2539 2541 2543 2545 2547 2549 2551 H1H18095B 2553 2555 2557 2559 2561 2563 2565 2567 H1H18096B 2569 2571 2573 2575 2577 2579 2581 2583 H1H18097B 2585 2587 2589 2591 2593 2595 2597 2599 H1H18098B 2601 2603 2605 2607 2609 2611 2613 2615 H1H18099B 2617 2619 2621 2623 2625 2627 2629 2631 H1H18100B 2633 2635 2637 2639 2641 2643 2645 2647 H1H18101B 2649 2651 2653 2655 2657 2659 2661 2663 H1H18102B 2665 2667 2669 2671 2673 2675 2677 2679 H1H18103B 2681 2683 2685 2687 2689 2691 2693 2695 H1H18104B 2697 2699 2701 2703 2705 2707 2709 2711 H1H18105B 2713 2715 2717 2719 2721 2723 2725 2727 H1H18107B 2729 2731 2733 2735 2737 2739 2741 2743 H1H18108B 2745 2747 2749 2751 2753 2755 2757 2759 H1H18109B 2761 2763 2765 2767 2769 2771 2773 2775 H1H18110B 2777 2779 2781 2783 2785 2787 2789 2791 H1H18111B 2793 2795 2797 2799 2801 2803 2805 2807 H1H18112B 2809 2811 2813 2815 2817 2819 2821 2823 H1H18113B 2825 2827 2829 2831 2833 2835 2837 2839 H1H18114B 2841 2843 2845 2847 2849 2851 2853 2855 H1H18115B 2857 2859 2861 2863 2865 2867 2869 2871 H1H18116B 2873 2875 2877 2879 2881 2883 2885 2887 H1H18117B 2889 2891 2893 2895 2897 2899 2901 2903 H1H18118B 2905 2907 2909 2911 2913 2915 2917 2919 H1H18119B 2921 2923 2925 2927 2929 2931 2933 2935 H1H18120B 2937 2939 2941 2943 2945 2947 2949 2951 H1H18121B 2953 2955 2957 2959 2961 2963 2965 2967 H1H18122B 2969 2971 2973 2975 2977 2979 2981 2983 H1H18123B 2985 2987 2989 2991 2993 2995 2997 2999 H1H18124B 3001 3003 3005 3007 3009 3011 3013 3015 H1H18125B 3017 3019 3021 3023 3025 3027 3029 3031 H1H18126B 3033 3035 3037 3039 3041 3043 3045 3047 H1H18127B 3049 3051 3053 3055 3057 3059 3061 3063 H1H18128B 3065 3067 3069 3071 3073 3075 3077 3079 H1H18129B 3081 3083 3085 3087 3089 3091 3093 3095 H1H18130B 3097 3099 3101 3103 3105 3107 3109 3111 H1H18131B 3113 3115 3117 3119 3121 3123 3125 3127 H1H18132B 3129 3131 3133 3135 3137 3139 3141 3143 H1H18133B 3145 3147 3149 3151 3153 3155 3157 3159 H1H18134B 3161 3163 3165 3167 3169 3171 3173 3175 H1H18135B 3177 3179 3181 3183 3185 3187 3189 3191 H1H18136B 3193 3195 3197 3199 3201 3203 3205 3207 H1H18137B 3209 3211 3213 3215 3217 3219 3221 3223 H1H18138B 3225 3227 3229 3231 3233 3235 3237 3239 H1H18139B 3241 3243 3245 3247 3249 3251 3253 3255 H1H18140B 3257 3259 3261 3263 3265 3267 3269 3271 H1H18141B 3273 3275 3277 3279 3281 3283 3285 3287 H1H18142B 3289 3291 3293 3295 3297 3299 3301 3303 H1H18143B 3305 3307 3309 3311 3313 3315 3317 3319 H1H18144B 3321 3323 3325 3327 3329 3331 3333 3335 H1H18145B 3337 3339 3341 3343 3345 3347 3349 3351 H1H18146B 3353 3355 3357 3359 3361 3363 3365 3367 H1H18147B 3369 3371 3373 3375 3377 3379 3381 3383 H1H18148B 3385 3387 3389 3391 3393 3395 3397 3399 H1H18149B 3401 3403 3405 3407 3409 3411 3413 3415 H1H18150B 3417 3419 3421 3423 3425 3427 3429 3431 H1H18151B 3433 3435 3437 3439 3441 3443 3445 3447 H1H18152B 3449 3451 3453 3455 3457 3459 3461 3463 H1H18153B 3465 3467 3469 3471 3473 3475 3477 3479 H1H18154B 3481 3483 3485 3487 3489 3491 3493 3495 H1H18155B 3497 3499 3501 3503 3505 3507 3509 3511 H1H18156B 3513 3515 3517 3519 3521 3523 3525 3527 H1H18157B 3529 3531 3533 3535 3537 3539 3541 3543 H1H18158B 3545 3547 3549 3551 3553 3555 3557 3559 H1H18159B 3561 3563 3565 3567 3569 3571 3573 3575 H1H18160B 3577 3579 3581 3583 3585 3587 3589 3591 H1H18161B 3593 3595 3597 3599 3601 3603 3605 3607 H1H18162B 3609 3611 3613 3615 3617 3619 3621 3623 H1H18163B 3625 3627 3629 3631 3633 3635 3637 3639 H1H18164B 3641 3643 3645 3647 3649 3651 3653 3655 H1H18165B 3657 3659 3661 3663 3665 3667 3669 3671 H1H18166B 3673 3675 3677 3679 3681 3683 3685 3687 H1H18167B 3689 3691 3693 3695 3697 3699 3701 3703 H1H18168B 3705 3707 3709 3711 3713 3715 3717 3719 H1H18169B 3721 3723 3725 3727 3729 3731 3733 3735 H1H18170B 3737 3739 3741 3743 3745 3747 3749 3751 H1H18171B 3753 3755 3757 3759 3761 3763 3765 3767 H1H18172B 3769 3771 3773 3775 3777 3779 3781 3783 H1H18173B 3785 3787 3789 3791 3793 3795 3797 3799 H1H18174B 3801 3803 3805 3807 3809 3811 3813 3815 H1H18175B 3817 3819 3821 3823 3825 3827 3829 3831 H1H18176B 3833 3835 3837 3839 3841 3843 3845 3847 H1H18177B 3849 3851 3853 3855 3857 3859 3861 3863 H1H18178B 3865 3867 3869 3871 3873 3875 3877 3879 H1H18179B 3881 3883 3885 3887 3889 3891 3893 3895 H1H18180B 3897 3899 3901 3903 3905 3907 3909 3911 H1H18181B 3913 3915 3917 3919 3921 3923 3925 3927 H1H18182B 3929 3931 3933 3935 3937 3939 3941 3943 H1H18183B 3945 3947 3949 3951 3953 3955 3957 3959 H1H18184B 3961 3963 3965 3967 3969 3971 3973 3975 H1H18185B 3977 3979 3981 3983 3985 3987 3989 3991 H1H18186B 3993 3995 3997 3999 4001 4003 4005 4007 H1H18187B 4009 4011 4013 4015 4017 4019 4021 4023 H1H18188B 4025 4027 4029 4031 4033 4035 4037 4039 H1H18189B 4041 4043 4045 4047 4049 4051 4053 4055 H1H18190B 4057 4059 4061 4063 4065 4067 4069 4071 H1H18191B 4073 4075 4077 4079 4081 4083 4085 4087 H1H18192B 4089 4091 4093 4095 4097 4099 4101 4103 H1H18193B 4105 4107 4109 4111 4113 4115 4117 4119 H1H18194B 4121 4123 4125 4127 4129 4131 4133 4135 H1H18195B 4137 4139 4141 4143 4145 4147 4149 4151 H1H18196B 4153 4155 4157 4159 4161 4163 4165 4167 H1H18197B 4169 4171 4173 4175 4177 4179 4181 4183 H1H18198B 4185 4187 4189 4191 4193 4195 4197 4199 H1H18199B 4201 4203 4205 4207 4209 4211 4213 4215 H1H18200B 4217 4219 4221 4223 4225 4227 4229 4231 H1H18201B 4233 4235 4237 4239 4241 4243 4245 4247 H1H18202B 4249 4251 4253 4255 4257 4259 4261 4263 H1H18203B 4265 4267 4269 4271 4273 4275 4277 4279 H1H18204B 4281 4283 4285 4287 4289 4291 4293 4295 H1H18205B 4297 4299 4301 4303 4305 4307 4309 4311 H1H18206B 4313 4315 4317 4319 4321 4323 4325 4327 H1H18207B 4329 4331 4333 4335 4337 4339 4341 4343 H1H18208B 4345 4347 4349 4351 4353 4355 4357 4359 H1H18209B 4361 4363 4365 4367 4369 4371 4373 4375 H1H18210B 4377 4379 4381 4383 4385 4387 4389 4391 H1H18211B 4393 4395 4397 4399 4401 4403 4405 4407 H1H18212B 4409 4411 4413 4415 4417 4419 4421 4423 H1H18213B 4425 4427 4429 4431 4433 4435 4437 4439 H1H18214B 4441 4443 4445 4447 4449 4451 4453 4455 H1H18216B 4457 4459 4461 4463 4465 4467 4469 4471 H1H18217B 4473 4475 4477 4479 4481 4483 4485 4487 H1H18218B 4489 4491 4493 4495 4497 4499 4501 4503 H1H18219B 4505 4507 4509 4511 4513 4515 4517 4519 H1H18220B 4521 4523 4525 4527 4529 4531 4533 4535 H1H18221B 4537 4539 4541 4543 4545 4547 4549 4551 H1H18222B 4553 4555 4557 4559 4561 4563 4565 4567 H1H18223B 4569 4571 4573 4575 4577 4579 4581 4583 H1H18224B 4585 4587 4589 4591 4593 4595 4597 4599 H1H18225B 4601 4603 4605 4607 4609 4611 4613 4615 H1H18226B 4617 4619 4621 4623 4625 4627 4629 4631 H1H18227B 4633 4635 4637 4639 4641 4643 4645 4647 H1H18228B 4649 4651 4653 4655 4657 4659 4661 4663 H1H18229B 4665 4667 4669 4671 4673 4675 4677 4679 H1H18230B 4681 4683 4685 4687 4689 4691 4693 4695 H1H18231B 4697 4699 4701 4703 4705 4707 4709 4711 H1H18232B 4713 4715 4717 4719 4721 4723 4725 4727 H1H18233B 4729 4731 4733 4735 4737 4739 4741 4743 H1H18234B 4745 4747 4749 4751 4753 4755 4757 4759 H1H18235B 4761 4763 4765 4767 4769 4771 4773 4775 H1H18236B 4777 4779 4781 4783 4785 4787 4789 4791 H1H18237B 4793 4795 4797 4799 4801 4803 4805 4807 H1H18238B 4809 4811 4813 4815 4817 4819 4821 4823 H1H18239B 4825 4827 4829 4831 4833 4835 4837 4839 H1H18240B 4841 4843 4845 4847 4849 4851 4853 4855 H1H18241B 4857 4859 4861 4863 4865 4867 4869 4871 H1H18242B 4873 4875 4877 4879 4881 4883 4885 4887 H1H18243B 4889 4891 4893 4895 4897 4899 4901 4903 H1H18244B 4905 4907 4909 4911 4913 4915 4917 4919 H1H18245B 4921 4923 4925 4927 4929 4931 4933 4935 H1H18246B 4937 4939 4941 4943 4945 4947 4949 4951 H1H18247B 4953 4955 4957 4959 4961 4963 4965 4967 H1H18248B 4969 4971 4973 4975 4977 4979 4981 4983 H1H18249B 4985 4987 4989 4991 4993 4995 4997 4999 H1H18250B 5001 5003 5005 5007 5009 5011 5013 5015 H1H18251B 5017 5019 5021 5023 5025 5027 5029 5031 H1H18252B 5033 5035 5037 5039 5041 5043 5045 5047 H1H18253B 5049 5051 5053 5055 5057 5059 5061 5063 H1H18254B 5065 5067 5069 5071 5073 5075 5077 5079 H1H18255B 5081 5083 5085 5087 5089 5091 5093 5095 H1H18256B 5097 5099 5101 5103 5105 5107 5109 5111 H1H18257B 5113 5115 5117 5119 5121 5123 5125 5127 H1H18258B 5129 5131 5133 5135 5137 5139 5141 5143 H1H18259B 5145 5147 5149 5151 5153 5155 5157 5159 H1H18261B 5161 5163 5165 5167 5169 5171 5173 5175 H1H18262B 5177 5179 5181 5183 5185 5187 5189 5191 H1H18263B 5193 5195 5197 5199 5201 5203 5205 5207 H1H18264B 5209 5211 5213 5215 5217 5219 5221 5223 H1H18265B 5225 5227 5229 5231 5233 5235 5237 5239 H1H18266B 5241 5243 5245 5247 5249 5251 5253 5255 H1H18267B 5257 5259 5261 5263 5265 5267 5269 5271 H1H18268B 5273 5275 5277 5279 5281 5283 5285 5287 H1H18269B 5289 5291 5293 5295 5297 5299 5301 5303 H1H18270B 5305 5307 5309 5311 5313 5315 5317 5319 H1H18271B 5321 5323 5325 5327 5329 5331 5333 5335 H1H18272B 5337 5339 5341 5343 5345 5347 5349 5351 H1H18274B 5353 5355 5357 5359 5361 5363 5365 5367 H1H18275B 5369 5371 5373 5375 5377 5379 5381 5383 H1H18276B 5385 5387 5389 5391 5393 5395 5397 5399 H1H18277B 5401 5403 5405 5407 5409 5411 5413 5415 H1H18278B 5417 5419 5421 5423 5425 5427 5429 5431 H1H18279B 5433 5435 5437 5439 5441 5443 5445 5447 H1H18280B 5449 5451 5453 5455 5457 5459 5461 5463 H1H18281B 5465 5467 5469 5471 5473 5475 5477 5479 H1H18282B 5481 5483 5485 5487 5489 5491 5493 5495 H1H18283B 5497 5499 5501 5503 5505 5507 5509 5511 H1H18284B 5513 5515 5517 5519 5521 5523 5525 5527 H1H18285B 5529 5531 5533 5535 5537 5539 5541 5543 H1H18286B 5545 5547 5549 5551 5553 5555 5557 5559 H1H18287B 5561 5563 5565 5567 5569 5571 5573 5575 H1H18288B 5577 5579 5581 5583 5585 5587 5589 5591 H1H18289B 5593 5595 5597 5599 5601 5603 5605 5607 H1H18290B 5609 5611 5613 5615 5617 5619 5621 5623 H1H18291B 5625 5627 5629 5631 5633 5635 5637 5639 H1H18292B 5641 5643 5645 5647 5649 5651 5653 5655 H1H18293B 5657 5659 5661 5663 5665 5667 5669 5671 H1H18294B 5673 5675 5677 5679 5681 5683 5685 5687 H1H18295B 5689 5691 5693 5695 5697 5699 5701 5703 H1H18297B 5705 5707 5709 5711 5713 5715 5717 5719 H1H18298B 5721 5723 5725 5727 5729 5731 5733 5735 H1H18299B 5737 5739 5741 5743 5745 5747 5749 5751 H1H18300B 5753 5755 5757 5759 5761 5763 5765 5767 H1H18301B 5769 5771 5773 5775 5777 5779 5781 5783 H1H18302B 5785 5787 5789 5791 5793 5795 5797 5799 H1H18303B 5801 5803 5805 5807 5809 5811 5813 5815 H1H18304B 5817 5819 5821 5823 5825 5827 5829 5831 H1H18305B 5833 5835 5837 5839 5841 5843 5845 5847 H1H18306B 5849 5851 5853 5855 5857 5859 5861 5863 H1H18307B 5865 5867 5869 5871 5873 5875 5877 5879 H1H18308B 5881 5883 5885 5887 5889 5891 5893 5895 H1H18309B 5897 5899 5901 5903 5905 5907 5909 5911 H1H18310B 5913 5915 5917 5919 5921 5923 5925 5927 H1H18311B 5929 5931 5933 5935 5937 5939 5941 5943 H1H18312B 5945 5947 5949 5951 5953 5955 5957 5959 H1H18313B 5961 5963 5965 5967 5969 5971 5973 5975 H1H18314B 5977 5979 5981 5983 5985 5987 5989 5991 H1H18315B 5993 5995 5997 5999 6001 6003 6005 6007 H1H18316B 6009 6011 6013 6015 6017 6019 6021 6023 H1H18317B 6025 6027 6029 6031 6033 6035 6037 6039 H1H18318B 6041 6043 6045 6047 6049 6051 6053 6055 H1H18319B 6057 6059 6061 6063 6065 6067 6069 6071 H1H18320B 6073 6075 6077 6079 6081 6083 6085 6087 H1H18321B 6089 6091 6093 6095 6097 6099 6101 6103 H1H18322B 6105 6107 6109 6111 6113 6115 6117 6119 H1H18323B 6121 6123 6125 6127 6129 6131 6133 6135 H1H18324B 6137 6139 6141 6143 6145 6147 6149 6151 H1H18325B 6153 6155 6157 6159 6161 6163 6165 6167 H1H18326B 6169 6171 6173 6175 6177 6179 6181 6183 H1H18327B 6185 6187 6189 6191 6193 6195 6197 6199 H1H18328B 6201 6203 6205 6207 6209 6211 6213 6215 H1H18329B 6217 6219 6221 6223 6225 6227 6229 6231 H1H18330B 6233 6235 6237 6239 6241 6243 6245 6247 H1H18331B 6249 6251 6253 6255 6257 6259 6261 6263 H1H18332B 6265 6267 6269 6271 6273 6275 6277 6279 H1H18333B 6281 6283 6285 6287 6289 6291 6293 6295 H1H18334B 6297 6299 6301 6303 6305 6307 6309 6311 H1H18335B 6313 6315 6317 6319 6321 6323 6325 6327

Example 9. H1H11729P has a Superior Pharmacokinetic Profile in Mice and Non-Human Primates (NHPs) Compared to a Comparator Antibody (Control I mAb)

A study was conducted to compare the pharmacokinetic profile of H1H11729P with a comparator antibody in mice and in non-human primates. One anti-influenza HA comparator antibody designated herein as Control I mAb, is an anti-influenza HA antibody with heavy (HC) and light chain (LC) amino acid sequences as set forth in WO2008/028946 as SEQ ID NO: 65 (HC) and SEQ ID NO: 91 (LC) and is also referred to in WO2008/028946 as CR6261.

For both the mouse and NHP PK experiments, circulating drug levels were determined by total human antibody analysis using an ELISA immunoassay. Briefly, a goat anti-human IgG polyclonal antibody was coated onto 96-well plates to capture the tested human antibodies in the sera, and then plate bound antibodies were detected using a goat anti-human IgG polyclonal antibody conjugated with horseradish and TMB substrate. The serum samples were in six-dose serial dilutions and reference standards of the respective antibodies in 12-dose serial dilutions. Drug antibody concentrations in the sera were calculated based on the reference standard curve generated using Graphpad Prism software.

A. Mouse Study

The pharmacokinetic assessment of H1H11729P was conducted in wild-type (WT) C57BL/6 mice. H1H11729P and Control I mAb were administered SC in separated groups of 5 mice each at a dose of 1 mg/kg. Bleeds were collected post-injection at 6 h, 1, 2, 3, 4, 7, 11, 14, 22, and 30 days in addition to the bleed collected one day prior to the antibody injection (pre-bleed). Serum fractions from the bleeds were separated and frozen at −80° C. until analysis was conducted.

Results:

As shown in Table 14, the half-life of H1H11729P in WT mice was 11.1 days, while the half-life of Control I mAb was 5.67 days, demonstrating an advantageous property of the H1H11729P over the comparator antibody.

TABLE 14 Summary of the Pharmacokinetic Profiles of H1H11729P and Control I mAb in Uninfected Wild-Type Mice Drug T_(1/2) (d) Cmax (μ/mL) AUC(d* μ/mL) H1H11729P 11.1 ± 0.8 12.0 ± 0.9  147 ± 3.4 Control I mAb 5.67 ± 0.6 10.4 ± 0.6 93.8 ± 4.8 B. Cynomolgus NHPs

Assessment of the pharmacokinetic clearance rate of H1H11729P and Control I mAb was conducted in 3-5 year old, female cynomolgus monkeys. Monkeys were prescreened for influenza A and B antibodies and were found to be negative. The antibody was tested in three animals and was administered subcutaneously at a dose of 3 mg/kg (at a volume of 2 mL/kg and 1.5 mg/mL). Blood samples were collected prior to dosing and post-injection at 1 h, 4 h, 8 h, and on Days 1, 2, 3, 4, 5, 10, 14, 18, 21, 24, 28, 35, 42, 49, 56, 63, 70, 84, and 98. Serum was separated from whole blood and frozen at −80° C. until analysis. Circulating drug concentrations were determined by total human antibody analysis using an ELISA immunoassay. Drug antibody concentrations in the sera were calculated based on the reference standard curve generated using Graphpad Prism software.

Results:

As shown in Table 15, the half-life of H1H11729P in monkeys was 13.4 days, while the half-life of Control I mAb was 8.05 days, demonstrating an advantageous property of the H1H11729P over the comparator antibody.

TABLE 15 Summary of the Pharmacokinetic Profiles of H1H11729P and Control I mAb in Cynomolgus Monkeys Drug T_(1/2) (d) Cmax (μg/mL) AUC(d* μg/mL) H1H11729P 13.4 ± 1.46 41.3 ± 7.84 863 ± 145  Control I mAb 8.05 ± 2.17 24.9 ± 1.87 320 ± 70.1

Example 10: H1H11729P Effectively Treats Lethal Influenza Virus Infection in Mice

There is a substantial unmet need for improved standard of care therapies for treating or preventing of influenza virus infections in humans. Currently, only two classes of drugs are available: the adamantanes and the neuraminidase inhibitors (NAIs). Adamantanes (amantadine and rimantadine) have been associated with the rapid emergence of drug-resistant strains and are no longer recommended for treatment of influenza. NAIs like oseltamivir (TAMIFLU®) are the front line drugs for treatment and prophylaxis of influenza, however, their window of efficacy is limited: NAIs have been shown to reduce the duration of fever and illness symptoms by about one day in the therapeutic setting if the antiviral is administered within 48 hours of symptom onset with little clinical evidence for efficacy if administered after 48 hours.

To evaluate the in vivo efficacy of H1H11729P in the treatment of severe influenza, experiments were conducted with the following objectives:

Study 1: To evaluate the efficacy of a single dose of H1H11729P versus the clinical standard of care oseltamivir (TAMIFLU®) given twice per day for 5 days.

Study 2: To determine the efficacy of H1H11729P administered in combination with oseltamivir.

The strain used in these studies included a historical [A/Puerto Rico/08/1934 (H1N1)] influenza A virus group 1 isolate. All experiments were performed in 6-week-old wild-type (BALB/c) female mice. Mice were challenged with 10×mouse LD₅₀ (MLD₅₀) equivalent to 800 plaque-forming units (PFUs) of A/Puerto Rico/08/1934 (H1N1). In the treatment models, mice were challenged intranasally (IN) on day 0 post-infection (p.i.) and fixed doses of mAb were given intravenously (IV) on specific days post-infection (e.g., day 1, 2, 3, 4 or 5). Oseltamivir was resuspended according to the manufacturers instructions and mice were dosed every 12 h (i.e., twice per day; BID) via oral gavage for 5 days, with the first dose administered on day 2 or 3 post-infection. Mice were weighed and observed daily up to day 14 p.i. and were sacrificed when they lost 20% of their starting weight. Results are reported as percent survival.

In the first experiment the efficacy of either a single dose of H1H11729P (15 mg/kg) or 25 or 5 mg/kg of oseltamivir BID (5 day regimen) initiating 48 hours post-infection (ie. administered therapeutically) was studied to evaluate the effect in the murine model at this timepoint. All mice receiving 5 mg/kg oseltamivir died by day 6, while the dose of 25 mg/kg improved survival to 40%; in contrast, all mice receiving H1H11729P survived (FIG. 1 and Table 16).

In the next experiment, H1H11729P was tested in combination with oseltamivir, beginning 72 hours post-infection. While only approximately 20% of mice treated with 25 mg/kg oseltamivir survived, single doses of H1H11729P at 15 mg/kg and 7 mg/kg resulted in 60% and 36% survival, respectively. An additive effect was observed when the treatments were combined: A single dose of 7 mg/kg H1H11729P combined with a regimen of oseltamivir showed 60% survival and a single dose of 15 mg/kg H1H11729P in combination with a regimen of oseltamivir resulted in 87% survival (FIG. 2 and Table 17).

In summary, H1H11729P displayed robust efficacy in treating mice infected with a severe historical influenza strain, and in fact, demonstrated greater efficacy than oseltamivir at 48 hours post infection. Furthermore, H1H11729P demonstrated additive efficacy when administered in combination with oseltamivir.

TABLE 16 Results of Study 1: A Single Dose of H1H11729P at 48 hrs p.i. Demonstrates Greater Efficacy than oseltamivir at 48 hrs p.i. in Treating Severe Influenza A Virus Infection in Mice Percent survival Number (no. of surviving of mice mice/total no. of PID per group mice in the group) Oral gavage control (uninfected) 5 100 (5/5) H1H11729P 5 100 (5/5) Oseltamivir (25 mg/kg BID × 5 d) 5 40 (2/5) Oseltamivir (5 mg/kg BID × 5 d) 5 0 (0/5) hIgG1 negative isotype control 5 0 (0/5) Oral gavage control (infected) 5 0 (0/5)

TABLE 17 Results of Study 2: Additive Efficacy is Observed when a Single Dose of H1H11729P is Combined with oseltamivir at 72 hrs p.i. to Treat Severe Influenza in Mice Percent survival Number (no. of surviving of mice mice/total no. of PID per group mice in the group) H1H11729P (15 mg/kg) 15 60 (9/15) H1H11729P (7 mg/kg) 15 35.7 (5/15) H1H11729P (15 mg/kg) + 15 86.7 (13/15) Oseltamivir (25 mg/kg BID × 5 d) H1H11729P (7 mg/kg) + 15 60 (9/15) Oseltamivir (25 mg/kg BID × 5 d) Oseltamivir (25 mg/kg BID × 5 d) 15 20 (3/15) hIgG1 negative isotype control 15 0 (0/15) Oral gavage control (infected) 15 0 (0/15) 

What is claimed is:
 1. An isolated antibody or antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 50; and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:
 66. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising a HCVR that comprises an amino acid sequence set forth in SEQ ID NO:
 50. 3. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising a LCVR that comprises an amino acid sequence set forth in SEQ ID NO:
 66. 4. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising a HCVR that comprises an amino acid sequence set forth in SEQ ID NO: 50; and a LCVR that comprises an amino acid sequence set forth in SEQ ID NO:
 66. 5. The isolated antibody or antigen-binding fragment of claim 4 which is an isolated antibody.
 6. The isolated antibody of claim 5 which is a recombinant antibody.
 7. The isolated antibody of claim 5 which is bispecific.
 8. A pharmaceutical composition comprising the isolated antibody of claim 5 and a pharmaceutically acceptable carrier or diluent.
 9. The pharmaceutical composition of claim 8, further comprising a second therapeutic agent.
 10. The pharmaceutical composition of claim 9, wherein the second therapeutic agent is an anti-viral drug.
 11. The pharmaceutical composition of claim 9, wherein the second therapeutic agent is a neuraminidase inhibitor or an interferon.
 12. The pharmaceutical composition of claim 9, wherein the second therapeutic agent is zidovudine, gangcyclovir, vidarabine, idoxuridine, trifluridine, foscarnet, acyclovir, ribavirin, amantadine, remantidine, saquinavir, indinavir, ritonavir, zanamivir, oseltamivir, laninamivir, peramivir or rimantadine.
 13. The pharmaceutical composition of claim 9, wherein the second therapeutic agent is an antibody that specifically binds to a protein other than hemagglutinin.
 14. A polynucleotide molecule comprising a polynucleotide sequence that encodes a HCVR, or an LVCR, or both, of the isolated antibody of claim
 5. 15. A vector comprising the polynucleotide molecule of claim
 14. 16. An isolated cell comprising the vector of claim
 15. 17. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising: (a) a HCDR1 that comprises an amino acid sequence set forth in SEQ ID NO: 52; (b) a HCDR2 that comprises an amino acid sequence set forth in SEQ ID NO: 54; (c) a HCDR3 that comprises an amino acid sequence set forth in SEQ ID NO: 56; (d) a LCDR1 that comprises an amino acid sequence set forth in SEQ ID NO: 68; (e) a LCDR2 that comprises an amino acid sequence set forth in SEQ ID NO: 70; and (f) a LCDR3 that comprises an amino acid sequence set forth in SEQ ID NO:
 72. 